28-DAY AMMONIA TOXICITY TESTING OF NON-PULMONATE SNAILS

EPA Contract Number: EP-C-09-001

Work Assignment 2-31, Task 1, Subtask 1

Final Report

PREPARED FOR THE

U.S. Environmental Protection Agency

By

Great Lakes Environmental Center

1295 King Ave.

Columbus, OH 43212

Phone: (614) 487-1040

Facsimile: (614) 487-1920

May 27, 2011
ii

TABLE OF CONTENTS

EXECUTIVE SUMMARY ........................................................................................................... vi

INTRODUCTION .......................................................................................................................... 1

OBJECTIVE ................................................................................................................................... 2

METHODS ..................................................................................................................................... 3

Experimental Facility .................................................................................................................. 3

Dilution Water............................................................................................................................. 3

Test Material ............................................................................................................................... 4

Test Organisms............................................................................................................................ 4

Toxicity Range Finding Test....................................................................................................... 4

28- Day Toxicity Test ................................................................................................................. 5

Data Analysis .............................................................................................................................. 6

RESULTS AND DISCUSSION..................................................................................................... 6

Toxicity Range Finding Test....................................................................................................... 6

28-Day Toxicity Test – Water Quality........................................................................................ 7

28-Day Toxicity Test - TAN Measurement and Concentrations................................................ 8

Determination of a 96-h EC50 in the 28-day Exposure.............................................................. 9

Determination of EC20s............................................................................................................ 10

TEST UNCERTAINTY................................................................................................................ 10

Variation in TAN concentrations and select water quality parameters..................................... 11

Data interpretation..................................................................................................................... 12

SUMMARY AND CONCLUSIONS ........................................................................................... 13

REFERENCES ............................................................................................................................. 14
iii

LIST OF TABLES

Table 1. Water quality summary statistics for 96-hr ammonia toxicity range-finding test with 

mature, mixed-age P. canaliculata. .............................................................................................. 15

Table 2. Results of the 96-hr range-finding test with mature, mixed-age P. canaliculata (N=6 

snails per treatment)...................................................................................................................... 16

Table 3. Overall water quality summary statistics for 28-day ammonia toxicity test with mature, 

mixed-age P. canaliculata. Values represent summary statistics derived using all measured 

values within a treatment (nominal) for a given parameter. ......................................................... 17

Table 4. Daily average measured total ammonia concentrations (mg TAN/L) by nominal 

treatment level. Number in parentheses following the coefficient of variation (CV) is the number 

of available TAN measurements at that concentration on that day, i.e., N = 1 to 4 based on 

number of replicates sampled per treatment. A value of (0) indicates that the TAN concentration 

for that day was estimated by interpolating the average concentrations from the closest days for 

which measurements were taken (see text)................................................................................... 18

Table 5. Daily average calculated unionized ammonia concentration (mg UIA/L) by nominal 

treatment level. Number in parentheses following the coefficient of variation (CV) is the number 

of available measurements at that concentration on that day, i.e., N = 1 to 4 based on number of 

replicates sampled per treatment. A value of (0) indicates that the UIA concentration for that day 

was estimated by interpolating the average concentrations from the closest days for which 

measurements were taken (see text). ............................................................................................ 19

Table 6. Concentration-response results for P. canaliculata 28 day survival in response to TAN, 

with and without replicates from nominal 1.9 mg TAN/L treatment. Toxicity predictions 

obtained from a 3-parameter logistic model using the EPA TRAP (v. 1.21) analysis program... 20

LIST OF FIGURES

Figure 1. TRAP logistic equation results of 96-hr range-finding test at pH 8.19 and 24.7°C. 

Ninety-six hour EC50 normalized to pH 8.0 and 25°C is 13.56 (9.91-18.53) mg TAN/L. TAN 

concentration represents average concentration calculated from the measurement of samples 

taken over the 96-hr test period. Statistics below figure represent output from TRAP where 

“initial” represents the input value required as a first guess to generate a model and where “final” 

represents actual statistic given by the fitted model equation....................................................... 21
iv

Figure 2. Graphical display of the variability in measured water temperatures over the 28-day 

test period amongst replicate chambers within the various test treatments (based on nominal 

TAN concentrations)..................................................................................................................... 22

Figure 3. Graphical display of the variability in measured pH of test water over the 28-day test 

period amongst replicate chambers within the various test treatments (based on nominal TAN 

concentrations).............................................................................................................................. 23

Figure 4. Graphical display of the variability in measured dissolved oxygen concentrations over 

the 28-day test period amongst replicate chambers within the various test treatments (based on 

nominal TAN concentrations)....................................................................................................... 24

Figure 5. Graphical display of the variability in measured TAN concentrations over the 28-day 

test period amongst replicate chambers within the various test treatments (based on nominal 

TAN concentrations). Line represents the daily average TAN from all replicates within a 

treatment when measured, and interpolation between the daily average concentrations when 

measurements were not made. ...................................................................................................... 25

Figure 6. Graphical display of the variability in calculated unionized ammonia (UIA) 

concentrations over the 28-day test period amongst replicate chambers within the various test 

treatments (based on nominal TAN concentrations). Line represents the daily average UIA from 

all replicates within a treatment when measured, and interpolation between the daily average 

concentrations when measurements were not made. .................................................................... 26

Figure 7. Twenty eight day survival of P. canaliculata across an ammonia concentration 

gradient with replicate values averaged. P. canaliculata in the control treatment were 

maintained for an additional 14 days after test completion to further assess controls. ................ 27

Figure 8. Twenty-eight day survival of P. canaliculata across an ammonia concentration gradient 

by replicate chamber. Note the anomalous early mortality in replicates 3 and 4 of the nominal 

1.9 mg TAN/L treatment............................................................................................................... 28

Figure 9. Graphical display of concentration-response results for P. canaliculata after 28 days in 

response to TAN, (a) all replicates from all treatments included, and (b) minus the four replicates 

from the second lowest (nominal 1.9 mg TAN/L) treatment. TRAP logistic equation results are 

as those presented in Table 5. ....................................................................................................... 29

LIST OF APPENDICES

APPENDIX 1. Justification for selection of test species and species distribution. ..................... 30
v

APPENDIX 2. Summary of acquisition, acclimation, maintenance, and health of test snail 

species........................................................................................................................................... 35

APPENDIX 3. Metals characterization of dilution water from East Arm Grand Traverse Bay, 

Lake Michigan. ............................................................................................................................. 42

APPENDIX 4. Cross validation of archived samples using new standard curve........................ 43

APPENDIX 5A. Water quality measurements. ........................................................................... 44

APPENDIX 6. TAN measurements............................................................................................. 50

APPENDIX 7. Trimatrix duplicate TAN analysis using Phenate Method.................................. 51

APPENDIX 8A. P. canaliculata survival.................................................................................... 52

APPENDIX 9. P. canaliculata growth measurements. ............................................................... 54
vi

EXECUTIVE SUMMARY

The purpose of the study outlined in this report was to independently test and confirm previous 

28-day toxicity test results indicating that specialized and/or Federally-Listed non-pulmonate, 

gill-bearing snails such as the Idaho springsnail, Bliss Rapids snail and pebblesnail are

potentially both: 1) extra sensitive to prolonged ammonia exposure, and 2) as sensitive as 

ammonia-sensitive freshwater unionid mussel species are to such exposure. To do so, EPA 

sponsored a study to conduct additional 28-day toxicity tests using more widely-distributed nonpulmonate snail species. The candidate non-pulmonate snail species targeted for testing were

mature, mixed-age globe siltsnail (Birgella subglobosus), pointed campeloma (Campeloma 

decisum), and silty hornsnail (Pleurocerca canaliculata), each representative of three widelydistributed families within the continental United States; Hydrobiidae, Viviparidae, and 

Pleuroceridae, respectively. Of the three wild-caught snail species collected for testing, EPA’s 

contractor (Great Lakes Environmental Center, Inc., i.e., GLEC) was only able to successfully 

hold, maintain, and test, via a 28-day, measured flow-through ammonia toxicity test, silty 

hornsnail (P. canaliculata). The 96 h acute EC50 estimated from a toxicity range finding test 

with the species was 13.56 mg total ammonia nitrogen or TAN/L, after normalizing to 25°C and 

pH 8.0. The corresponding EC20 estimated from the 28-day survival test was as low as 0.55 mg 

TAN/L after normalizing to 25°C and pH 8.0; however, because of the uncertainty in the test, the 

recommended 28-day ammonia survival effects concentration for the species is <2.28 mg TAN/L 

at 25°C and pH 8.0. This upper limit concentration is not recommended for FCV calculation as 

originally intended, but instead, should be considered as contributing to the weight of evidence 

indicating the unique sensitivity of gill-bearing snails to extended ammonia exposure in general. 

An ACR estimated by dividing the normalized 96 h EC50 estimate from the range finding test by 

the normalized and recommended 28-day ammonia survival effects concentration is calculated to 

be >5.9.

Based on the results reported herein, GLEC concludes that for P. canaliculata, the 28-day

ammonia survival effects concentration of <2.28 mg TAN/L at 25°C and pH 8.0 confirms that:

1) The sensitivity of mature, mixed age P. canaliculata to ammonia toxicity via a 28-day

test approaches that of their more specialized and/or Federally-Listed counterparts, e.g., 

Idaho springsnail (Pyrgulopsis idahoensis), Bliss Rapids (Taylorconcha serpenticola)

and pebblesnails (Fluminicola sp.) – see Besser et al. 2009 and Besser (2011); and,

2) May also approach that of the highly ammonia-sensitive freshwater unionid mussel 

species: wavy-rayed lamp mussel, Lampsilis fasciola; fatmucket, Lampsilis siliquoidea; 

and rainbow mussel, Villosa iris – see Wang et al. (2007a, b) and Wang et al. (2008).

Additional 28-day toxicity tests are recommended for this and other non-pulmonate snails to 

further substantiate the preliminary findings obtained in this and other similar studies with 

freshwater gill-bearing snails.
1

INTRODUCTION

The U.S. EPA, Office of Water is charged with protecting aquatic life, wildlife, and human 

health from adverse anthropogenic and water-mediated effects under the purview of the Clean 

Water Act (CWA). In support of this mission, on December 30, 2009, U.S. EPA’s Health and 

Ecological Criteria Division (HECD), within the Office of Science and Technology (OST) of 

EPA, published the “Draft 2009 Update Aquatic Life Ambient Water Quality Criteria for 

Ammonia – Freshwater” in the Federal Register (FR). Included in the 2009 draft ammonia 

criteria update are several 28-day EC20 values (concentrations associated with a 20% effect) 

from a series of toxicity tests with various freshwater snail species from a United States 

Geological Survey (USGS) study (Besser et al. 2009). The Besser et al. (2009) study involved 

the conduct of 28-day flow-through survival and growth toxicity tests with five species of snails: 

four non-pulmonate or gill-bearing snails (Pyrgulopsis idahoensis, Fontigens aldrichi, 

Taylorconcha serpenticola, and Fluminicola sp.), and one commonly tested pulmonate (capable 

of air-breathing) snail (Lymnaea stagnalis). Two of the non-pulmonate species were FederallyListed species at the time of testing, i.e., P. idahoensis and T. serpenticola, and all of the nonpulmonate snails tested require specialized habitats and exhibit limited distribution within the 

continental United States.

All snail tests performed in the Besser et al. (2009) study were conducted in ASTM hard water 

(mean hardness and alkalinity of 169 and 121 mg/L as CaCO3, respectively) which had a pH 

range of 8.20-8.29 and a temperature range of 19-21oC during testing. The pH (8.0) and 

temperature (25oC) normalized 28-day EC20 (based on survival and expressed in terms of total 

ammonia nitrogen (TAN)) for the five snail species ranged from a low of <0.52 mg TAN/L for 

juvenile Idaho springsnail (P. idahoensis) to >8.5 mg TAN/L for less than 1 week old post-hatch 

pond snails (L. stagnalis). The 28-day EC20 for the other three non-pulmonate snail species was 

lower than the 28-day EC20 for L. stagnalis, i.e., the 28-day EC20 for the mixed age Ozark 

springsnail (F. aldrichi) was 0.71 mg TAN/L; 3.98 mg TAN/L for the mixed age Bliss Rapids 

snail (T. serpenticola); 3.77 mg TAN/L for the adult Idaho springsnail (P. idahoensis); and <7.9 

mg TAN/L for the mixed age pebblesnail (Fluminicola sp.). Thus, it appears in this instance that 

gill-bearing, non-pulmonate snail species may be slightly to substantially more sensitive to 28-

day ammonia exposure than at least one non-gill bearing, pulmonate snail species, L. stagnalis. 

Moreover, the comparatively low survival-based 28-day EC20 for the juvenile Idaho springsnail 

coincides with the Species Mean Chronic Values (SMCVs) reported in the 2009 draft ammonia 

criteria update for several ammonia-sensitive freshwater mussel species (e.g., wavy-rayed lamp 

mussel, Lampsilis fasciola; fatmucket, Lampsilis siliquoidea; and rainbow mussel, Villosa iris) –

see Wang et al. (2007a, b) and Wang et al. (2008). Therefore, EPA commissioned GLEC to 

conduct additional 28-day toxicity tests targeting more widely-distributed non-pulmonate snail 

species to evaluate whether this group of snail species is generally: 1) as sensitive to 28-day

ammonia toxicity as their more specialized and/or Federally-Listed counterparts (e.g., 

springsnail, Bliss Rapids snail and pebblesnail), and 2) as sensitive as ammonia-sensitive 

freshwater mussel species.
2

OBJECTIVE

The specific objective of this study was to generate 28-day toxicity test data to determine the 

concentration of TAN that causes 20 percent lethality and/or immobilization to one or more 

widely-distributed snail species within the continental United States. Candidate non-pulmonate 

snail species were identified using species distributions from NatureServe©Explorer. GLEC, 

working in cooperation with U.S. EPA, selected three candidate snail species based on the 

following factors: 1) distribution within the conterminous United States, 2) degree of habitat 

specialization, 3) representativeness of the taxonomic group as a whole, and 4) availability for 

collection (see Appendix 1). Of the three species collected for testing: siltsnail (Birgella 

subglobosus), pointed campeloma (Campeloma decisum), and silty hornsnail (Pleurocerca 

canaliculata), only the latter species was successfully acclimated and maintained at a level of 

health necessary for 28-day ammonia toxicity testing (see Appendix 2 for additional details 

regarding the acquisition and maintenance of all three snail species targeted for testing, and for a 

short summary of the health status achieved during their acclimation and holding in the 

laboratory). 

The 28-day ammonia toxicity test data for P. canaliculata described herein are to be used to 

inform the final revision of EPA’s update of the freshwater ambient water quality criteria for the 

protection of aquatic life (ALC) for ammonia. The 28-day toxicity tests were performed 

generally following applicable guidance outlined in ASTM E 1241-05, Standard Guide for 

Conducting Early Life-Stage Toxicity Tests with Fishes (ASTM 2010a), and supplemented with 

any applicable methods and protocol established in ASTM E2455-06, Standard Guide for 

Conducting Laboratory Toxicity Tests with Freshwater Mussels (ASTM 2010b). As aquatic 

toxicity tests utilizing non-pulmonate snails are extremely limited, acclimation and holding 

procedures were also based on other laboratory experience (e.g., the Besser et al. (2009) study 

conducted at the USGS facility in Columbia, MO) gained from 28-day snail toxicity testing, and 

following recommendations from snail culture experts such as Paul Johnson at Alabama Aquatic 

Biodiversity Center. It is important to recognize the current knowledge of snail culturing, 

holding and feeding is very limited and not completely applicable to aquatic toxicity testing. 

Due to certain limitations such as the inability to culture the selected snail species to obtain test 

organisms of a certain life stage (e.g., young juveniles), the primary focus for this study was on 

testing survival of mature, mixed age snails. And because mature, mixed age snails were 

targeted for use, growth over the 28-day test period was expected to be slow and thus, this 

endpoint was not expected to provide useful concentration-response information. Instead, the 

study conducted by GLEC focused on survival/immobility as the endpoint for 28-day point 

estimation/effect determination. 

To provide additional ancillary information potentially useful for ALC development for 

ammonia, the test protocol included enhanced observations of live/dead counts and collection of 

samples for chemical analysis to support calculation of a 96-hr EC50. Prior to testing, a 96-hr 

acute toxicity range-finding test was used to establish the test concentrations for the 28-day 

toxicity tests. The acute EC50 estimated from the 96-hr range-finding test and 28-day ammonia 
3

survival effects concentration1were subsequently used to estimate an Acute-Chronic Ratio 

(ACR) for P. canaliculata.

METHODS

Experimental Facility

The 28-day toxicity test was conducted using a modified Benoit semi-continuous flow minidilutor system at GLEC’s environmental toxicology laboratory in Traverse City, Michigan. The 

dilutor system was located in an enclosed facility with a dedicated water source and electricity. 

Dilution water used in this study was collected from Lake Michigan, delivered to an external 

temperature-controlled storage tank that in turn delivered water to a single internal glass headbox 

within the enclosed facility. Dilution water from the glass headbox was then gravity fed to the 

mini-dilutor system. All construction materials for this system consisted of conditioned glass, 

PVC, or TYGONTM, and 100% clear silica gel was used as the adhesive material.

Dilution water in both the internal headbox and external storage tank was continuously aerated 

and mixed. Water temperature within the external storage tank was maintained at 25±1°C with 

an electronic heater/chiller unit. Water within the internal headbox was maintained at 25°C 

through a combination of temperature control within the facility coupled with immersion heaters 

as required. The facility is equipped with an array of lights that were maintained at 150-500 lux 

under a photoperiod of 16 hours light and 8 hours dark. 

An ammonia stock solution was continuously maintained throughout the study, and this stock 

solution was continuously distributed to a mixing cell within the mini-dilutor that continuously 

received a constant supply of Lake Michigan dilution water and ammonia stock solution via a 

metered pump (Fluid Metering, Inc.). The resulting solution was then distributed to the first 

treatment mixing cell of the system, representing the highest ammonia test concentration. A 

portion of this test solution was then used to create subsequent two-fold serial dilutions for the 

four remaining treatment mixing cells resulting in five ammonia concentrations with an 

approximate dilution factor of 0.5. The test solution within each treatment level mixing cell was 

then evenly distributed to the four replicate 2.5 L test chambers for the corresponding treatment 

level via flow-splitting boxes. The mini-dilutor system was monitored daily and cleaned and 

calibrated as required to maintain a flow rate of approximately 32 mL/min from the outlets of 

mixing cells that were positioned in-line with and directly above the six flow-splitting boxes 

resulting in a volume of water sufficient to provide at least 3.5 complete test solution renewals 

per replicate test chamber per day. 

Dilution Water

The laboratory dilution water for the acclimation period and toxicity test was Lake Michigan 

water that was collected directly from the West Arm of Grand Traverse Bay and delivered to

 

1 Note: GLEC recommends use of the 28-day ammonia survival effects concentration for this test instead of an

EC20 point estimate because of the uncertainty in the 28-day test due to variable ammonia exposure, particularly in 

the first 18 days of the test, and varying response within critical treatments – see additional explanation under Test 

Uncertainty below.
4

GLEC’s laboratory. Similar dilution water has previously been characterized for: pH, total 

suspended solids, total organic carbon (TOC), organophosphorus pesticides, total residual 

chlorine, total PCBs, chlorinated phenoxy herbicides, ammonia, cyanide, sulfide, bromide, 

fluoride, iodide, nitrate, phosphate, sulfate, calcium, magnesium, potassium, aluminum, arsenic, 

beryllium, boron, cadmium, chromium, cobalt, copper, iron, lead, manganese, mercury, 

molybdenum, nickel, selenium, silver, zinc, hardness, alkalinity, conductivity, sodium and 

chloride. The characterization (shown for metals and other select constituents only) is 

summarized in Appendix 3. In every case, the concentration of the analyzed components in the 

dilution water is less than the concentration shown to adversely affect freshwater aquatic 

invertebrate species based on current 304(a) ambient water quality criteria and datasets, or is 

below the minimum detection limit. 

The dilution water was pH adjusted and controlled to maintain a prescribed test pH using an 

automated pH controller (BarnantTM) in the internal headbox and adjusting the pH of the dilution 

water with the addition of dilute HCl.

Test Material

A mixture of reagent grade ammonium chloride (NH4Cl: BDH) and ammonium hydroxide 

(NH4OH: EM Science) was used in this 28-day toxicity test to more effectively maintain a 

constant pH in the test solutions. Stock solutions to be delivered to the dilutor system were 

initially prepared as aqueous solutions in de-ionized water. Prior to test initiation, the 

appropriate mixture of NH4Cl and NH4OH to maintain a target pH of 8.0 was identified.

Test Organisms

Mixed age adult Pleurocera canaliculata (Family Pleuroceridae) were collected from the Ohio 

River on August 11, 2010 (see Appendix 2 for additional details regarding location, water quality 

conditions, acclimation procedures, etc.). Snails were transported from the collection site to the 

Traverse City, MI experimental facility in cool, oxygenated Ohio River water. Upon arrival at 

the experimental facility the following morning, snails were transferred to Lake Michigan test 

dilution water (see Appendix 2 for further detail) and held until September 22, 2010 

(approximately six weeks total) at approximately 25°C and pH=8.1 to acclimate snails to test 

water and conditions as well as to ensure wild caught organisms did not show any signs of stress, 

physical damage, disease, external parasites, or any other condition that would contribute to 

mortality. 

Snails were fed a diet of periphyton ad libitum on unglazed PVC tiles introduced initially and 

after approximately two weeks into each test chamber during the study, and were also observed 

supplementing that diet by consuming periphyton biofilms that accrued on test chamber surfaces, 

particularly during the latter stages of the experiment. Periphyton was colonized in a separate 

flow through system receiving Lake Michigan dilution water by allowing periphyton biofilms to 

colonize and develop on the unglazed PVC tiles.

96-hr Toxicity Range-Finding Test

Prior to the initiation of the 28-day toxicity test, a 96-hr toxicity range-finding test was 

conducted to determine a suitable range of test concentrations for use in the 28-day toxicity test. 

The range-finding test ran from September 14-18, 2010, and consisted of five treatment levels 
5

and a control, with three adult snails per replicate and two replicates per treatment (N=6 snails 

total per treatment). Nominal treatment concentrations for the range-finding test were 3, 6, 12, 

25 and 50 mg TAN/L. Target test temperature and pH were 25°C and 8.0, respectively. The 

endpoint for this range-finding test was death and/or immobilization, which was measured daily 

and used to calculate a 96-hr EC50 for the test. Individuals presumed dead were removed from 

the test chamber and held in a separate beaker containing overlying water for up to 30 minutes, 

and if observations were confirmed, dead individuals were discarded. If death was not 

confirmed, then individuals were returned to their respective test chamber.

28- Day Toxicity Test

Following the completion of the range-finding test, a 28-day toxicity test using mature, mixedage Pleurocera canaliculata was conducted from September 22 – October 20, 2010. The test 

design consisted of five ammonia treatment levels and one control treatment. Each treatment 

consisted of four replicate chambers with six snails (N=24 snails total per treatment). Note: for 

this study, only 280 P. canaliculata were collected from the Ohio River, 46 of which were 

smaller in size (range: 0.5 to 0.8 cm total shell length) and subsequently not used for 28-day 

toxicity testing. Because of the expedited timeline for this study, and also because of the length 

of time chosen a priori to acclimate and hold the wild-caught snails prior to testing to ensure 

adequate health (approximately six weeks), sample size could not be increased to the desired 

number of ten without causing a delay in testing. Nominal treatment concentrations were 0.9, 

1.9, 3.8, 7.5, and 15 mg TAN/L. The target test temperature and pH were 25°C and 8.0, 

respectively. The endpoint for the 28-day toxicity test was death and/or immobilization, which 

was measured daily and used to attempt calculation of a definitive EC50 for the first 96 hours of 

the test and an EC20 for the 28 day test period2. Except for the first 96 hours of the 28-day test 

where observations were made daily, dead and/or immobile snails were evaluated on Mondays, 

Wednesdays and Fridays of each week, as described above for the range-finding test.

GLEC experienced some difficulty maintaining the desired target water quality conditions 

throughout the test largely due to problems with the maintenance and operation of the dilutor 

system under the difficult test conditions (i.e., high test temperature and use of natural, unfiltered 

lake water) selected for testing (see additional details provided in the Test Uncertainty section 

provided below). Dissolved oxygen, temperature and pH were measured daily in each replicate 

chamber on nearly every day of the testing period using bench top and hand held meters:

dissolved oxygen [YSI Model 57 or 52]; pH [Orion Model 710A or 250A]; temperature [VWR 

Traceable Thermometer-digital]). Specific conductivity was measured weekly in each replicate 

on nearly every Monday of the testing period using a bench top meter (YSI Model 35). The 

minimum acceptable dissolved oxygen concentration in any test chamber was 60% saturation, 

and any time dissolved oxygen fell below 70% saturation, the replicate chamber in question was 

further investigated and the problem remedied, primarily through increased attention and 

maintenance of the mini-dilutor system. Likewise, the pH of the test water within a given test 

chamber was maintained to within 0.2 standard units of the target pH of 8.0, and temperature to 

within ± 1°C of the target temperature of 25°C. If any individual measured pH or temperature in 

any test chamber was more than 1.5 pH units or 3

oC (respectively) different from the mean of the 

 

2 Again, due to uncertainty in the test, GLEC recommends an alternate 28-day ammonia survival effects 

concentration for the test, as described in more detail in the Test Uncertainty section of this report.
6

time-weighted average measured temperature or pH for the individual test chambers, then the 

replicate chamber in question was further investigated and the problem remedied, again, 

primarily through increased attention and maintenance of the mini-dilutor system. If all snails 

within a particular test chamber died, then dissolved oxygen, temperature, and pH measurements 

for that chamber were no longer taken.

TAN measurements were measured at GLEC with an Orion Research Microprocessor pH 

millivolt meter 811 with an Orion 9512 ion specific ammonia probe throughout the study and 

within replicate test chambers following U.S. EPA method 350.3, Standard Methods for the 

Examination of Water and Wastewater, 21st edition, 2005, Method 4500-NH3 (D) and 

corresponding GLEC Standard Operating Procedures (SOPs). The reporting limit for this 

method was 0.5 mg TAN/L. Ammonia measurements were made in all chambers on days 1, 2, 

5, 16, 19, and 21-28 of the test, the latter (days 21-28 of the test) only in test chambers with 

surviving snails. On the remaining test days, ammonia concentrations were measured in a 

minimum of two replicate chambers per treatment every other day. In addition to the metered 

measurements, a subsample of 35 measurements from water samples collected between days 20 

and 28 were analyzed for TAN by a second laboratory (Trimatrix) using the Phenate Method 

(Standard Methods for the Examination of Water and Wastewater, 21st edition, 2005, Method 

4500-NH3 (G)). Subsamples of ten archived samples were measured after the study at GLEC 

but with a re-calculated standard curve with a lower concentration range (see Appendix 4).

One day prior to test initiation, the metering system and flow rates upstream and out of the flowsplitting boxes were checked to assure the system was functioning properly. The meter system 

and flow rates were subsequently checked daily throughout the remainder of the 28-day test 

period. Water from all replicate chambers was siphoned and chambers were allowed to fill 

overnight with test water. A total of six adult P. canaliculata were randomly assigned to each 

test chamber from a single holding chamber. Snails used in the range-finding test were not used 

in the 28-day test. Water quality measurements and observations of survival were made 

throughout the study as previously described, with sufficient measurements made during the first 

96 hours to determine a 96-hr EC50. After the completion of the 28-day test, all surviving snails 

from the control group were held for an additional 14 days in control water and monitored for 

survival or immobilization to further demonstrate the health of the snails used in this test.

Data Analysis

All effect concentration (EC20 and EC50) measurements were made using three parameter 

logistic equation functions within EPA’s Toxicity Relationship Analysis Program (TRAP, 

Version 1.21, October 30, 2010). The survival NOEC and LOEC of the 28-day toxicity test were 

not calculated for the test for reasons explained later in the report. Summary statistics for all 

water quality parameters (including TAN concentrations) were calculated in MS Excel.

RESULTS AND DISCUSSION

96-hr Toxicity Range-Finding Test

Average temperature, pH, and dissolved oxygen throughout the toxicity range-finding test were 

similar across treatments, with temperature ranging from 24.3-25.1°C, pH ranging from 8.07-

8.23, and dissolved oxygen ranging from 7.3-7.6 mg/L (Table 1). The overall average 
7

temperature (24.7°C) was similar to the target temperature of 25°C, while the overall average pH 

(8.19) was slightly higher than the target value of 8.0. Mean measured TAN concentrations were 

on average 36 percent lower than nominal concentrations across nearly the entire concentration 

range except for the lowest concentration (Table 1). Overall, measured TAN increased over the 

course of the range-finding test, possibly due to improved performance and operation of the 

dilutor and ammonia delivery system. 

Prior to the initiation of the 28-day toxicity test, a 96-hr EC50 estimate was calculated from the 

results of the range finder test using the EPA Probit Analysis Program (see Table 2). Because 

the control concentrations were below detection level, they were not included in the initial 

analysis. The resulting 96-hr EC50 at measured test conditions was 9.06 mg TAN/L, with a 95% 

confidence interval of 6.00-15.0 mg TAN/L (Table 2). After normalizing to pH 8.0 and 25°C, 

the normalized 96-hr EC50 was 12.71 mg TAN/L, with a 95% confidence interval of 8.42-21.1

mg TAN/L.

The 96-hr EC50 was recalculated in TRAP using a logistic equation model after assigning a 

concentration of 0.06 mg TAN/L to the controls (see text below under 28-Day Toxicity Test –

TAN Measurement and Concentrations). Based on measured test conditions (pH-8.19, Temp24.7°C) the resulting 96-hr EC50 was 9.66 mg TAN/L, with a 95% confidence interval of 7.06-

13.2 mg TAN/L (Figure 1). After normalizing to pH 8.0 and 25°C, the 96-hr EC50 was 

estimated to be 13.56 mg TAN/L, with a 95% confidence interval of 9.91-18.53 mg TAN/L, but 

because of low sample size (N=6 per concentration) in the range-finding test, this particular 96-

hr EC50 should be considered preliminary for this species.

28-Day Toxicity Test – Water Quality

Temperature, pH, and dissolved oxygen levels fluctuated throughout the study, particularly over 

the 4-day period from Friday, October 1, 2010 to Monday October 4, 2010, test days 9-12 (see 

Figures 2, 3 and 4, respectively; corresponding raw data for each treatment is included in 

Appendix 5). During this period, several measured values for water temperature, pH, and 

dissolved oxygen were reduced relative to the remainder of the test period. The lowest 

temperatures during this period fell just below the acceptable range (<22oC) for this test (Figure 

2). Similarly, the lowest pH values observed were just below 7.5 (Figure 3), and dissolved 

oxygen fell below the acceptable range (60% saturation) in a total of five test chambers (Figure 

4). Further explanation for the likely decrease in these key water quality parameters is provided 

in the Test Uncertainty section below.

Overall, the average water temperature, pH, and dissolved oxygen levels were 24.7oC, 8.15, and 

7.3 mg/L, respectively. Average water temperature for a given treatment in general fell just 

below the target temperature of 25oC throughout the entire test concentration (range of 24.5 to 

24.8oC), while pH was slightly higher overall (average difference of approximately 0.07 s.u.) at 

the lower end of the ammonia concentration gradient (including the control treatment) than at the 

high end, possibly as a result of the relative ratios of NH4Cl to NH4OH across treatments (Table 

3). Mean dissolved oxygen concentrations ranged from a low of 7.1 mg/L in the 1.9 and 3.8 mg 

TAN/L (nominal) treatments to an average high of 7.7 mg/L in the control treatment (Table 3). 

Specific conductivity was relatively stable throughout the study within a given ammonia 

treatment level; however, specific conductivity increased from 307-415 μS/cm across the 
8

concentration gradient, most likely the result of the additional chloride from the test material 

(Table 3).

28-Day Toxicity Test - TAN Measurement and Concentrations

The temporal distribution of TAN measurements taken during the test was uneven, with more 

measurements taken during test days 1-5 (to determine a definitive 96-hr EC5) and 19-28 (to 

assess the effectiveness of the remedial actions taken to address the problems GLEC encountered 

trying to maintain stable TAN concentrations within each treatment), respectively, and fewer 

measurements taken between days 6-18 (Figure 5; Appendix 6). Because there was a high 

degree of temporal variability in observed TAN concentrations, average TAN for a given 

treatment cannot be accurately estimated as a simple average of all measurements within a 

particular treatment. This is because the average TAN concentration for a particular treatment 

will be biased towards the more data rich sampling periods, which do not encompass the full 

range of temporal variability during the 28-day study. In order to remove this potential source of 

bias from the average TAN measurements, the following procedure was performed. For every 

day during the test, the average daily TAN concentration was calculated for each treatment level 

(Table 4). By collapsing the replicate measurements into a single treatment level average on a 

daily basis, much of the problem associated with the paucity of measurement data during days 6-

18 is overcome. While TAN may not have been measured in a single replicate chamber for up to 

a week (or longer, in those chambers that had already experienced 100% snail mortality), TAN 

was measured in at least one, and usually at least two chambers, on most test days (see Appendix 

6). For those days where no measurements were taken in any replicate chamber for a particular 

treatment level, the daily average was estimated through interpolation of the nearest temporal 

measurements available. For example, in the nominal 1.9 mg TAN/L treatment, there were no 

measurements in any of the replicates on test day 6. The estimated concentration of 0.53 mg

TAN/L is the average of the day 5 concentration of 0.50 mg TAN/L and the day 7 concentration 

of 0.56 mg TAN/L. It is worth noting that the average daily concentrations estimated using this 

method are generally lower than the concentrations one would obtain from averaging all of the 

available measurements, because for all but the highest TAN treatment level, the data are most 

depauperate during the period when they are also the lowest (Figure 5). This approach allows 

for the cumulative average TAN concentrations to be calculated for the entire 28-day test period

which are used to calculate the 28-day EC20s described later in this report.

Based on the above, the 28-day cumulative average TAN concentrations (i.e., treatment mean 

TAN concentrations, or x ̄ in Table 4) were below nominal concentrations for all treatment levels 

(Table 4). Mean measured TAN as a percentage of nominal TAN increased across the 

concentration gradient, rising from 38% at the lowest test concentration to 80% at the highest test 

concentration. TAN concentrations were temporally variable in all test concentrations, with the 

ratio of highest to lowest measured TAN concentration for single measurements within each 

treatment all exceeding 1.5 (Figure 5; also see Appendix 6). The relative temporal fluctuations 

in TAN measurements were highest in the two lowest test concentrations, peaking in the second 

lowest test concentration (nominal = 1.9 mg TAN/L; measured = 0.81 mg TAN/L), then 

decreasing with increasing test concentration for the three highest treatment levels (Figure 5, 

Table 4). Note that within the three test concentrations representing the middle of the 

concentration gradient (e.g., measured 0.81, 1.86 and 4.50 mg TAN/L), the lowest measured 

TAN concentration coincided with test days 9-12, which was the timeframe when temperature, 
9

pH and dissolved oxygen also dropped below target test specifications (compare Figure 5 against 

Figures 2 through 4). Also note that the highest measured TAN concentration for all but the 

highest treatment level generally occurred on the first day of the 28-day test. These withintreatment fluctuations in TAN measurements amongst replicate test chambers explain a 

significant portion of the variability in exposure for the test, and thus, are a major source of 

uncertainty precluding accurate estimation of the 28-day EC20, as discussed in greater detail 

below. 

For convenience to the reader, the temporal distribution of calculated unionized ammonia (UIA) 

concentrations was summarized in table form and graphically as for TAN (see Table 5 and 

Figure 6). The pattern of temporal variability observed for TAN is similarly evident for UIA, but 

even more pronounced.

Due to the observed variability in measured TAN concentrations, and as an attempt to not only 

verify the accuracy and precision of the metered TAN measurements but also to check the 

validity of the TAN measurements, as many as 35 additional water samples were collected

between days 20-28 for TAN measurement and a corresponding split sample was sent to a

second laboratory and analyzed for TAN using the phenate method (Method 4500-NH3 (G)). 

The phenate method was more sensitive by an order of magnitude than the meter method 

(reporting limit 0.05 mg TAN/L). The seven sub-samples from the experimental control were all 

below the phenate method reporting limit of 0.05 mg TAN/L. Concentrations of the non-control 

samples measured by the phenate method were approximately 1.3 times higher than 

corresponding metered measurements, and that relative difference was similar across the test 

concentration gradient (Appendix 7).

As part of the remedial action taken above, an additional ten sub-samples were also collected 

during days 20-28 and archived by GLEC until the completion of the study. After the 

completion of the study, the sub-samples were re-measured by the same meter used for the study, 

but using a refined standard curve equation with additional standards at the low end of the 

concentration gradient. Using the revised standard curve, the cross-validated control 

concentration was 0.06 mg TAN/L (Appendix 4).

As can be seen in Table 4 and corresponding appendix (Appendix 6), and in order to facilitate 

calculation of 28-day EC20s using TRAP, all non-detects were assigned the value of 0.06 mg 

TAN/L obtained from re-measuring archived samples using the revised standard curve as 

described above. The value of 0.06 mg TAN/L was likely a conservative estimate, based on the 

results of the seven control sub-samples measured using the phenate method, all of which were 

below the method reporting limit of 0.05 mg TAN/L. 

Determination of a 96-hr EC50 in the 28-Day Exposure

After 96 hours, P. canaliculata survival was greater than 50 percent for all test concentrations in 

the 28-day exposure. As a result, a 96-hr EC50 could not be calculated from the data generated 

during the 28-day exposure using either TRAP or the Probit Analysis Program. Similarly, 

ANOVA analysis of 96-hr survival revealed no statistically significant differences between 

treatments (P=0.3), so no post hoc analysis was performed. The 96-hr acute EC50 based on the 

first four days of the 28-day toxicity test was >9.59 mg TAN/L after normalizing the highest 
10

TAN treatment test concentration measured after 96-hr (i.e., cumulative average of 9.83 mg 

TAN/L at pH 8.02 and temperature 24.2oC) to 25oC and pH 8.0. This is in contrast to the result 

from the acute range-finding test where a 96-hr EC50 of 13.56 mg TAN/L normalized to 25oC 

and pH 8.0 was determined for the species.

Determination of 28-Day EC20s

Viewed in the aggregate, snail mortality increased with increasing TAN concentration, and the 

initiation of snail mortality generally occurred sooner at higher TAN concentrations compared to 

lower concentrations (see Figures 7 and 8; and corresponding raw survival data provided in 

Appendix 8A and B). When individual replicates within each treatment are considered, 

however, it is apparent that replicates 3 and 4 within the second lowest treatment concentration 

(nominal – 1.9 mg TAN/L; measured – 0.81 mg TAN/L) experienced very rapid mortality, with 

only one surviving snail in each of these replicates after three days, and with replicate 3 

experiencing complete mortality after four days (Figure 8). The onset of mortality for the snails 

in these two replicate chambers occurred earlier than for snails in any of the other test 

concentrations, and was likely the result of some unmeasured factor aside from ammonia 

toxicity. Water quality measurements in those chambers during that time period provide little

additional evidence of why the rapid die-off may have occurred (see additional details in the Test 

Uncertainty section below). Moreover, in contrast to the early onset of mortality for snails in 

replicates 3 and 4 of this treatment, all six snails survived in replicate 1 (Figure 8). Because of 

the obvious incongruity in response amongst replicates within this treatment (nominal - 1.9 mg 

TAN/L; measured - 0.81 mg TAN/L), dose-response analysis was performed in TRAP on the 28-

day survival response of snails with (Figure 9a) and without (Figure 9b) the four replicates from 

this potentially anomalous treatment.

The 28-day EC20 estimate based on the response of snails in all replicates of every treatment at 

test conditions (pH 8.15 and 24.7°C) was 0.4475 mg TAN/L, with a 95% confidence interval of 

0.1657-1.208 mg TAN/L. Normalized to pH 8.0 and 25°C, the EC20 was 0.5508 mg TAN/L, 

with a 95% confidence interval of 0.2039-1.487 mg TAN/L (Table 6). In contrast, the 28-day 

EC20 estimate without the four replicates from the second lowest treatment at test conditions 

(pH 8.15 and 24.7°C) was 0.6841 mg TAN/L, with a 95% confidence interval of 0.2017-2.320

mg TAN/L. Normalized to pH 8.0 and 25°C, this EC20 was 0.8584 mg TAN/L, with a 95% 

confidence interval of 0.2531-2.911 mg TAN/L (Table 6).

There were no readily apparent differences amongst treatment groups based on measurements of 

growth taken on surviving snails at the termination of the test on day 28 (see Appendix 9), and 

therefore, growth data were not used for 28-day point estimation. 

TEST UNCERTAINTY

As is clear from above, a measure of uncertainty exists with this test owing to both the variable 

ammonia exposures and the unequal responses observed within and among treatments, such that 

the accuracy of the 28-day EC20 for the test is questionable. The following provides additional 

explanation regarding interpretation of these test results, as well as a recommendation on how 

these results might best be considered given this variability in exposures and responses.
11

Variation in TAN concentrations and select water quality parameters

As discussed in the preceding section, much of the temporal variability in TAN concentrations 

may be attributable to the very high TAN concentrations measured on test day 1 and very low 

TAN concentrations measured on test day 12. There is no obvious explanation for the high and 

low TAN measurements based upon review of test records and observations, with the exception 

of measured TAN stock concentrations. Meter calibration logs, recorded diluter flow rates 

measured in diluter cells upstream of the splitter boxes, ammonia stock preparation and 

procedure logs, etc., do not reveal any extraordinary circumstances over the particular timeframe 

in question (or throughout the entire 28-day test period).

A general decline in stock TAN concentration may partially explain some of the temporal 

variation in TAN concentration among treatments (see Table 4 and Appendix 6). However, 

records indicate that recipe and stock preparation procedures were implemented similarly

throughout the exposure; the exception being the small, likely inconsequential, addition of 

NH4OH (approximately 200 ml per 4 L) used to raise the pH of the stock solution to near 8.0. 

During the test, the measured TAN of freshly-prepared stock solutions declined over the first 12 

days by approximately 30 percent (422 to 295 mg TAN/L), after which ammonia stock 

concentrations increased to a maximum of 467 mg TAN/L on day 16 before stabilizing at a 

measured average concentration of 450 mg TAN/L for the remainder of the test (Note: fresh 

ammonia stock solutions were prepared daily or near daily from test days 21-28, but less were 

prepared every 2 to 5 days until then).

Corrective action to the problem of the declining (and variable) measured TAN concentrations 

among replicates and treatments included increased delivery system maintenance (e.g., scrubbing 

of flow-splitting boxes and adjustment and maintenance of siphons to ensure equal flows of test 

solution from flow-splitting boxes), as well as increased sampling for TAN measurement in 

every replicate chamber on every day over the final 10 days of the test. In addition, several 

duplicate samples were collected and sent to a third-party contract laboratory (Trimatrix) for 

analytical determination of ammonia using the Phenate Method (see text and Appendix 7). Also, 

additional samples taken from select replicates within each treatment and on select days were 

collected and measured for TAN throughout the day (i.e., to assess diurnal variability – note: as

these measured values were found to be consistent, the values were averaged for the purpose of 

reporting).

In addition to the 30% decline in TAN stock concentrations over the first 12 days of the 

experiment, snails within select replicates of all treatments did experience low water 

temperature, pH or dissolved oxygen (or any combination of the three) between test days 9 

through 12 (see Figures 2-4). Of these, only low dissolved oxygen could be considered to 

directly affect snail survival, but upon further review of these data, one cannot definitively 

conclude that the drop in dissolved oxygen resulted in decreased snail survival. For example, 

dissolved oxygen concentrations decreased to as low as 2.1 and 1.9 mg/L over the day 9 to 12 

timeframe in replicates 2 and 4 in the nominal 0.9 and 3.8 mg TAN/L treatments, respectively

(see Figure 4), but these decreases did not result in subsequent decreases in snail survival (see 

Figure 8 and Appendix 8). 
12

GLEC suspects the drop in temperature, pH, and dissolved oxygen between days 9-12, as well as 

some of the temporal variability in TAN concentrations between days 1-18, were the result of 

blocked flow from the diluter splitter boxes to select replicates (via line plugging by algae and 

other biological debris). Observations by laboratory technicians confirm such circumstance on 

several occasions, although any observations of no or reduced flow to test chambers was 

immediately rectified upon recognition by increased attention and proper maintenance (e.g., 

scrubbing or physical manipulation to remove blockages/debris or adjustment of siphons in flowsplitting boxes to re-establish equal flow to all test chambers). Nevertheless, with such low flow 

rates (only 3.5 turnovers per day) restriction of flow for even a short period of time could lead to 

large reductions in temperature, pH, dissolved oxygen and TAN concentration via ambient heat 

transfer (in the case of temperature) and bacterial and periphyton uptake and utilization (in the 

case of pH, dissolved oxygen and TAN). Note: the amount of periphyton and biofilm on the 

sides of individual exposure chambers was moderate to substantial and largely permitted (i.e., 

individual test chambers were only scrubbed on a weekly as opposed to a daily basis), as such 

material proved a supplemental food source to the snails during the test and was assumed to 

positively contribute to their overall health. This latter practice, in addition to the low turnover 

rates within each test chamber, was likely problematic as the dilution water used for testing was 

natural, unfiltered Lake Michigan water, and the test temperature was a high 25oC, thus 

promoting substantial periphyton growth within the test system.

Data interpretation

The variable TAN (and consequently, UIA) concentrations and unequal survival responses

among select replicates and treatments (see Figures 5, 6 and 7), particularly in the lower test 

concentrations, casts doubt on the reliability of associating effects with a particular "mean" TAN

concentration for this test. Temporal variability in TAN concentrations was the primary reason

GLEC calculated and reported cumulative daily average TAN concentration, which more closely 

reflects the average concentration experienced by snails over the course of the entire 28-day 

exposure period (data provided in Table 4). Despite the highly variable survival responses

within the nominal 1.9 mg TAN/L treatment (0.81 mg TAN/L measured), Figure 9a shows a 

relatively strong concentration-response relationship after 28 days. However, because of 

uncertainty associated with both the rapid mortality in two replicates as well as the overall high 

level inter-replicate variability within this treatment, it was decided that a more conservative 

approach would be to exclude this treatment completely from the analysis.

Nevertheless, given the above uncertainty, and given that the sample size was low for the test (N 

= 6 per replicate, 24 per treatment versus the ASTM recommended N=10 per replicate, 40 per 

treatment), a 28-day ammonia survival effects concentration is proposed in place of the 

calculated EC20 values reported in Table 6 of this report. The value of 1.86 mg TAN/L (28-day 

measured cumulative average concentration of the nominal 3.8 mg TAN/L exposure treatment) 

was selected based on the strong negative effect of ammonia on snail survival (67% reduction or 

more) over time at this and all higher test concentrations. The 28-day ammonia survival effects 

concentration, when normalized to 25oC and pH 8 assuming an average exposure temperature of 

24.7oC and pH 8.15, is 2.28 mg TAN/L. This effects concentration should be considered a 

maximum “unacceptable” 28-day survival effect concentration for the species above which

substantial mortality is expected. Owing to the uncertainty in this value, however, it is not 

recommended for FCV calculation as originally intended, but should instead be considered as 
13

contributing to the weight of evidence indicating the sensitivity of gill-bearing (non-Pulmonate) 

snails to prolonged ammonia exposure in general.

Finally, due to the low number of snails originally collected to support testing, GLEC was not 

able to repeat the test. However, GLEC advises that this test should be repeated in order to better 

define the 28-day survival EC20 for the species.

SUMMARY AND CONCLUSIONS

A 28-day ammonia toxicity test was completed with the non-pulmonate snail species Pleurocera 

canaliculata. Mature, mixed age snails were collected from the Ohio River and acclimated to 

test conditions for one month prior to testing. The 28-day survival EC20 for this snail species 

was determined to be as low as 0.55 mg TAN/L at pH 8 and 25oC; however, due to uncertainty 

in the point estimate, a 28-day ammonia survival effect concentration of <2.28 mg TAN/L 

representing a maximum “unacceptable” survival effect concentration is recommended instead. 

A corresponding ACR for the test is calculated as the quotient of the normalized 96-hr EC50 

estimated from the acute range-finding test (13.56 mg TAN/L) and the normalized 28-day 

ammonia survival effects concentration of <2.28 mg TAN/L, resulting in an ACR of >5.9.

Based on these results, GLEC concludes that for P. canaliculata, the low 28-d ammonia survival 

effects concentration of <2.28 mg TAN/L confirms that:

1) The sensitivity of mature, mixed age P. canaliculata to ammonia toxicity via a 28-day 

test approaches those of their more specialized and/or Federally-Listed counterparts (e.g., 

Idaho springsnail, Bliss Rapids and pebblesnails); and,

2) May also approach that of the highly ammonia-sensitive freshwater unionid mussel 

species: wavy-rayed lamp mussel, Lampsilis fasciola; fatmucket, Lampsilis siliquoidea; 

and rainbow mussel, Villosa iris – see Wang et al. (2007a, b) and Wang et al. (2008).

Additional 28-day toxicity tests are recommended for this and other non-pulmonate snails to 

further substantiate the preliminary findings obtained in this and other similar studies with 

freshwater gill-bearing snails.
14

REFERENCES

APHA. 1995. Standard Methods for the Examination of Water and Wastewater. 20th Edition. 

American Society for Testing and Materials. 1995. Standard Test Methods for Measuring the 

Toxicity of Sediment Associated Contaminants with Freshwater Invertebrates. E1706-95b. In 

Annual Book of Standards. Vol. 11.05. Philadelphia, PA. 

ASTM. 2010a. Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fish. 

E1241-05. In: Annual Book of Standards. Vol. 11.06. West Conshohocken, PA. 

ASTM. 2010b. Standard Guide for Conducting Laboratory Toxicity Tests with Freshwater 

Mussels. E2455-06. In Annual Book of ASTM Standards. Vol. 11.06. West Conshohocken, PA. 

Besser JM, Hardesty DL, Greer, IE, Ingersoll CG. 2009. Sensitivity of Freshwater Snails to 

Aquatic Contaminants: Chronic Toxicity of Copper, Ammonia and Pentachlorophenol to 

Endangered Snail Species and Surrogates. Report submitted to USEPA Duluth, MN by USGS, 

Columbia, MO (CERC-8335-FY07-20-10).

Newman MC. 1995. Advances in Trace Substance Research. Quantitative Methods in Aquatic 

Ecotoxicology. CRC Press, Inc., Boca Raton, FL. pp. 139-140.

United States Environmental Protection Agency. 1999. 1999 Update of ambient water quality 

criteria for ammonia. EPA-822-R-99-014. National Technical Information Service, Springfield, 

VA.

Wang N, Ingersoll CG, Hardesty DK, Ivey CD, Kunz JL, May TW, Dwyer FJ, Roberts AD, 

Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007a. Acute Toxicity of Copper, Ammonia, 

and Chlorine to Glochidia and Juveniles of Freshwater Mussels (Unionidae). Environ, Toxicol.

Chem. 26:2036-2047.

Wang N, Ingersoll CG, Greer IE, Hardesty DK, Ivey CD, Kunz JL, Brumbaugh WG, Dwyer FJ, 

Roberts AD, Augspurger T, Kane CM, Neves RJ, Barnhart MC. 2007b. Chronic Toxicity of 

Copper and Ammonia to Juvenile Freshwater Mussels (Unionidae). Environ. Toxicol. Chem.

26:2048-2056.

Wang N, Erickson RJ, Ingersoll CG, Ivey CD, Brunson EL, Augspurger T, Barnhart MC. 2008. 

Influence of pH on the Acute Toxicity of Ammonia to Juvenile Freshwater Mussels (Fatmucket, 

Lampsilis siliquoidea). Environ. Toxicol. Chem. 27:1141-1146.
15

Table 1. Water quality summary statistics for 96-hr ammonia toxicity range-finding test 

with mature, mixed-age P. canaliculata.

Statistic

Concentration

(mg TAN/L)

pH

Temp

(°C)

D.O.

Nominal Measured (mg/L)

Average Control 0.06 8.19 25.1 7.6

Std. Deviation n.a.a0.06 0.94 0.51

Minimum 0.06 8.11 23.7 6.8

Maximum 0.06 8.26 26.6 8.1

Average 3.0 2.62 8.25 24.7 7.5

Std. Deviation 1.64 0.08 1.0 0.46

Minimum 0.57 8.19 23.0 6.8

Maximum 4.10 8.47 26.3 8.1

Average 6.0 3.57 8.23 24.7 7.3

Std. Deviation 1.41 0.05 1.0 0.74

Minimum 2.05 8.15 23.6 6.4

Maximum 5.20 8.29 26.7 8.0

Average 12.0 7.64 8.19 24.3 7.3

Std. Deviation 2.04 0.07 0.87 0.34

Minimum 5.83 8.12 22.8 6.8

Maximum 10.2 8.30 25.3 7.7

Average 25.0 17.0 8.15 24.6 7.6

Std. Deviation 0.92 0.04 1.21 0.29

Minimum 15.9 8.10 23.0 7.1

Maximum 17.7 8.21 26.7 7.9

Average 50.0 32.8 8.07 24.7 7.5

Std. Deviation 3.52 0.06 0.96 0.66

Minimum 28.4 7.98 23.6 6.3

Maximum 36.1 8.17 26.4 8.0

a

All measurements were below detection limits. Concentrations set to 0.06 mg/L based on 

re-analysis of archived samples using revised standard curve equation (see text in Results 

and Discussion under 28-Day Toxicity Test – TAN Measurement and Concentrations).
16

Table 2. Results of the 96-hr range-finding test with mature, mixed-age P. canaliculata

(N=6 snails per treatment).

Nominal Concentration

(mg TAN/L)

Measured Concentration

(mg TAN/L)

P. canaliculata

Survival

(Number Alive out of 6)

Control 0.06 6

3.0 2.62 6

6.0 3.57 5

12 7.64 4

25 17.0 1

50 32.8 0

96 h EC50 (95% CI) 9.057 (5.996 – 15.042)a

a Calculated using EPA’s Probit Analysis Program (Version 1.5).
17

Table 3. Overall water quality summary statistics for 28-day ammonia toxicity test with

mature, mixed-age P. canaliculata. Values represent summary statistics derived 

using all measured values within a treatment (nominal) for a given parameter.

Statistic pH

Temp

(°C)

D.O.

(mg/L)

Specific

Conductance

(μS/cm)

Control

Average 8.22 24.8 7.7 307

Minimum 7.86 21.7 5.6 274

Maximum 8.73 26.1 9.0 324

0.90 mg TAN/L

Average 8.17 24.7 7.3 322

Minimum 7.43 21.2 2.1 311

Maximum 8.7 26.1 8.9 340

1.9 mg TAN/L

Average 8.14 24.6 7.1 330

Minimum 7.52 21.3 3.3 311

Maximum 8.64 26.2 10.1 354

3.8 mg TAN/L

Average 8.13 24.7 7.1 342

Minimum 7.27 21.8 1.9 317

Maximum 8.63 26.3 8.6 363

7.5 mg TAN/L

Average 8.18 24.7 7.6 361

Minimum 7.8 21.6 6.0 337

Maximum 8.67 26.3 9.4 387

15 mg TAN/L

Average 8.07 24.5 7.2 415

Minimum 7.43 21.7 3.3 399

Maximum 8.33 26.1 8.9 428
18

Table 4. Daily average measured total ammonia concentrations (mg TAN/L) by nominal treatment level. 

Number in parentheses following the coefficient of variation (CV) is the number of available TAN 

measurements at that concentration on that day, i.e., N = 1 to 4 based on number of replicates 

sampled per treatment. A value of (0) indicates that the TAN concentration for that day was 

estimated by interpolating the average concentrations from the closest days for which 

measurements were taken (see text).

Test

Day

Daily Average Measured TAN Concentration (mg TAN/L)

Control

Nominal 0.90 mg 

TAN/L

Nominal 1.9 mg 

TAN/L

Nominal 3.8 mg 

TAN/L

Nominal 7.5 mg 

TAN/L

Nominal 15 mg 

TAN/L

TAN CV TAN CV TAN CV TAN CV TAN CV TAN CV

1 0.06 0.00(4) 1.36 0.10(4) 2.57 0.11(4) 3.94 0.04(4) 7.15 0.02(4) 10.93 0.01(4)

2 0.06 0.00(4) 0.39 0.29(4) 0.88 0.28(4) 2.33 0.20(4) 4.06 0.04(4) 10.55 0.05(4)

3 0.06 0.00(2) 0.06 0.00(2) 0.74 0.03(2) 1.74 0.16(3) 3.69 0.03(2) 8.85 0.04(2)

4 0.06 na(0) 0.12 na(0) 0.62 na(0) 1.52 na(0) 3.58 na(0) 8.99 na(0)

5 0.06 0.00(4) 0.19 0.16(4) 0.50 0.02(3) 1.31 0.12(4) 3.47 0.05(4) 9.14 0.03(4)

6 0.06 na(0) 0.16 na(0) 0.53 na(0) 1.18 na(0) 3.49 na(0) 8.88 na(0)

7 0.06 0.00(2) 0.13 0.21(2) 0.56 0.03(2) 1.04 0.43(2) 3.50 0.03(2) 8.62 0.04(2)

8 0.06 na(0) 0.18 na(0) 0.68 na(0) 1.28 na(0) 4.18 na(0) 9.22 na(0)

9 0.06 0.00(2) 0.22 0.77(2) 0.79 0.08(2) 1.52 0.74(2) 4.86 0.03(2) 9.82 0.10(2)

10 0.06 na(0) 0.17 na(0) 0.55 na(0) 1.11 na(0) 3.75 na(0) 9.94 na(0)

11 0.06 na(0) 0.11 na(0) 0.30 na(0) 0.70 na(0) 2.64 na(0) 10.06 na(0)

12 0.06 0.00(2) 0.06 0.00(2) 0.06 0.00(2) 0.29 0.08(2) 1.53 0.06(2) 10.18 0.12(2)

13 0.06 na(0) 0.09 na(0) 0.39 na(0) 0.81 na(0) 3.44 na(0) 12.48 na(0)

14 0.06 0.00(2) 0.13 0.09(2) 0.71 na(1) 1.34 0.18(2) 5.36 0.02(2) 14.79 na(1)

15 0.06 na(0) 0.18 na(0) 0.76 na(0) 1.80 na(0) 5.54 na(0) 16.82 na(0)

16 0.06 0.00(4) 0.23 0.79(4) 0.81 0.55(3) 2.27 0.22(4) 5.72 0.02(4) 18.84 na(1)

17 0.06 na(0) 0.38 na(0) 1.10 na(0) 2.61 na(0) 6.00 na(0) 17.82 na(0)

18 0.06 na(0) 0.54 na(0) 1.39 na(0) 2.95 na(0) 6.27 na(0) 16.80 na(0)

19 0.06 0.00(4) 0.69 0.39(4) 1.69 0.40(4) 3.29 0.19(4) 6.54 0.07(4) 15.77 0.07(4)

20 0.06 na(0) 0.30 0.92(4) 1.07 0.13(3) 1.69 na(0) 4.78 na(0) 14.80 na(0)

21 0.06 0.00(4) 0.30 0.55(4) 0.93 0.21(3) 2.17 0.07(3) 4.78 0.03(3) 13.83 na(0)

22 0.06 0.00(4) 0.52 0.61(4) 0.69 0.56(3) 2.14 0.09(3) 4.56 0.02(3) 12.86 na(0)

23 0.06 0.00(4) 0.49 0.05(4) 0.97 0.49(3) 2.17 0.37(3) 5.16 0.04(3) 11.89 na(1)

24 0.06 0.00(4) 0.50 0.04(4) 0.79 0.57(3) 2.35 0.03(3) 4.44 0.05(2) 10.01 na(1)

25 0.06 0.00(4) 0.55 0.09(4) 0.66 0.95(3) 2.32 0.00(3) 4.49 0.04(2) 11.35 na(1)

26 0.06 0.00(4) 0.48 0.13(4) 0.49 1.21(3) 1.99 0.01(3) 4.17 0.06(2) 10.06 na(1)

27 0.06 0.00(4) 0.48 0.10(4) 0.76 0.34(3) 2.18 0.02(3) 4.51 0.04(2) 12.17 na(1)

28 0.06 0.00(4) 0.56 0.23(4) 0.76 0.54(3) 2.04 0.07(4) 4.26 0.07(4) 10.04 0.01(4)

Treatment Mean Concentration (mg TAN/L)a

x ̄ 0.06 0.34 0.81 1.86 4.50 11.98

Max 0.06 1.36 2.57 3.94 7.15 18.84

Min 0.06 0.06 0.06 0.29 1.53 8.62

a Treatment mean concentrations represent the 28-day average (x ̄ ) concentration. Maximum and minimum values represent the highest and 

lowest daily average concentration for a particular treatment level. The corresponding raw data of measured TAN for all replicates within 

treatments is provided in Appendix 6.
19

Table 5. Daily average calculated unionized ammonia concentration (mg UIA/L) by nominal treatment level. 

Number in parentheses following the coefficient of variation (CV) is the number of available 

measurements at that concentration on that day, i.e., N = 1 to 4 based on number of replicates 

sampled per treatment. A value of (0) indicates that the UIA concentration for that day was 

estimated by interpolating the average concentrations from the closest days for which measurements 

were taken (see text).

Test

Day

Daily Average UIA Concentration (mg NH3/L)

Control

Nominal 0.90 mg 

TAN/L

Nominal 1.9 mg 

TAN/L

Nominal 3.8 mg 

TAN/L

Nominal 7.5 mg 

TAN/L

Nominal 15 mg 

TAN/L

TAN CV TAN CV TAN CV TAN CV TAN CV TAN CV

1 0.0029 na(1) 0.0683 na(1) 0.1564 na(1) 0.2026 0.06(2) 0.4363 na(1) 0.5536 na(1)

2 0.0064 0.21(4) 0.0268 0.26(4) 0.0539 0.19(4) 0.1207 0.37(4) 0.3380 0.10(4) 0.7566 0.10(4)

3 0.0046 0.09(2) 0.0047 0.01(2) 0.0477 0.70(2) 0.1288 0.50(3) 0.2522 0.06(2) 0.5220 0.07(2)

4 0.0049 na(0) 0.0112 na(0) 0.0441 na(0) 0.1133 na(0) 0.2692 na(0) 0.5739 na(0)

5 0.0053 0.071(4) 0.0176 0.168(4) 0.0405 0.16(3) 0.0978 0.41(4) 0.2861 0.11(4) 0.6258 0.09(4)

6 0.0050 na(0) 0.0131 na(0) 0.0427 na(0) 0.1069 na(0) 0.2813 na(0) 0.5861 na(0)

7 0.0048 0.06(2) 0.0086 0.20(2) 0.0449 na(1) 0.1159 na(1) 0.2764 0.09(2) 0.5464 0.09(2)

8 0.0040 na(0) 0.0089 na(0) 0.0391 na(0) 0.0961 na(0) 0.2357 na(0) 0.4081 na(0)

9 0.0031 0.04(2) 0.0092 1.16(2) 0.0332 0.25(2) 0.0763 1.26(2) 0.1949 0.07(2) 0.2697 0.64(2)

10 0.0033 na(0) 0.0077 na(0) 0.0230 na(0) 0.0566 na(0) 0.1715 na(0) 0.4100 na(0)

11 0.0034 na(0) 0.0061 na(0) 0.0128 na(0) 0.0369 na(0) 0.1481 na(0) 0.5502 na(0)

12 0.0036 0.32(2) 0.0045 0.55(2) 0.0025 0.59(2) 0.0172 0.32(2) 0.1248 0.14(2) 0.6904 0.04(2)

13 0.0060 na(0) 0.0103 na(0) 0.0202 na(0) 0.0597 na(0) 0.4211 na(0) 1.1456 na(0)

14 0.0084 0.05(2) 0.0161 0.07(2) 0.0378 na(0) 0.1021 0.14(2) 0.7174 0.24(2) 1.6007 na(1)

15 0.0077 na(0) 0.0178 na(0) 0.0554 na(0) 0.1413 na(0) 0.7037 na(0) 1.5463 na(0)

16 0.0070 0.05(4) 0.0196 1.03(4) 0.0731 0.78(3) 0.1805 0.59(4) 0.6899 0.36(4) 1.4920 na(1)

17 0.0065 na(0) 0.0329 na(0) 0.0874 na(0) 0.2221 na(0) 0.7079 na(0)

18 0.0061 na(0) 0.0462 na(0) 0.1017 na(0) 0.2638 na(0) 0.7259 na(0)

19 0.0057 0.14(4) 0.0595 0.50(4) 0.1160 0.46(3) 0.3054 0.19(4) 0.7439 0.52(4)

20 0.0053 na(1) 0.0305 0.96(4) 0.1185 0.20(3) 0.1340 na(1) 0.6541 na(1)

21 0.0061 0.19(4) 0.0308 0.63(4) 0.0926 0.27(3) 0.2432 0.11(3) 0.4529 0.16(3)

22 0.0071 0.10(4) 0.0534 0.65(4) 0.0660 0.64(3) 0.1944 0.29(3) 0.4511 0.20(3)

23 0.0162 0.06(4) 0.1080 0.35(4) 0.2208 0.48(3) 0.4590 0.59(3) 1.0445 0.29(3)

24 0.0061 0.06(4) 0.0473 0.06(4) 0.0676 0.76(3) 0.2250 0.19(3) 0.4247 0.08(2)

25 0.0075 0.04(4) 0.0655 0.10(4) 0.0734 1.08(3) 0.2417 0.20(3) 0.3641 0.15(2)

26 0.0066 0.10(4) 0.0510 0.11(4) 0.0502 1.25(3) 0.2269 0.03(3) 0.2259 0.27(2)

27 0.0080 0.19(4) 0.0510 0.12(4) 0.0924 0.39(3) 0.2779 0.06(3) 0.4081 0.33(2)

28 0.0070 0.26(4) 0.0568 0.33(4) 0.0621 0.77(4) 0.2547 0.05(4) 0.2289 0.70(4)

Treatment Mean Concentration (mg UIA/L)a

x ̄ 0.0060 0.03156 0.06701 0.1679 0.4278 0.7673

Max 0.0162 0.1080 0.2208 0.4590 1.0445 1.6007

Min 0.0029 0.0045 0.0025 0.0172 0.1248 0.2697

a Treatment mean concentrations represent the 28-day average (x ̄ ) concentration. Maximum and minimum values represent the highest and 

lowest daily average concentration for a particular treatment level. The corresponding raw data used to calculate UIA concentrations for all 

replicates within treatments is provided in Appendices 5 (pH and temperature) and 6 (TAN concentrations), where UIA is calculated using the 

equations provided in the 1999 ambient water quality criteria document for ammonia- freshwater (EPA 1999).
20

Table 6. Concentration-response results for P. canaliculata 28 day survival in response to 

TAN, with and without replicates from nominal 1.9 mg TAN/L treatment. 

Toxicity predictions obtained from a 3-parameter logistic model using the EPA 

TRAP (v. 1.21) analysis program.

28 Day Dose-Response Results (all replicates every treatment)

Model Fit

Parameter Estimate Standard Error 95% LCL 95% UCL

Log(EC50) -0.06 -0.06 0.11 -0.30

Slope 1.19 1.19 0.53 0.10

Y-Intercept 0.91 0.91 0.11 0.68

Effect Concentrations (EC)

EC ECx 95% LCL 95% UCL

50 0.8750 0.5061 1.513

20 0.4475 0.1657 1.208

10 0.3023 0.0808 1.131

5 0.2106 0.0411 1.078

Normalized EC20 (pH=8, 25°C)

EC ECx 95% LCL 95% UCL

20 0.5508 0.2039 1.487

28 Day Dose-Response Results (minus replicates from nominal 1.9 mg TAN/L treatment) 

Model Fit

Parameter Estimate Standard Error 95% LCL 95% UCL

Log(EC50) 0.0942 0.12995 -0.17996 0.36838

Slope 1.3377 0.69655 -0.13189 2.8073

Y-Intercept 0.89187 0.0706 0.74297 1.0408

Effect Concentrations (EC)

EC ECx 95% LCL 95% UCL

50 1.2423 0.6608 2.336

20 0.6841 0.2017 2.320

10 0.4826 0.0984 2.367

5 0.3499 0.0505 2.423

Normalized EC20 (pH=8, 25°C)

EC ECx 95% LCL 95% UCL

20 0.8584 0.2531 2.911
21

Figure 1. TRAP logistic equation results of 96-hr range-finding test at pH 8.19 and 24.7°C. 

Ninety-six hour EC50 normalized to pH 8.0 and 25°C is 13.56 (9.91-18.53) mg 

TAN/L. TAN concentration represents average concentration calculated from the 

measurement of samples taken over the 96-hr test period. Statistics below figure 

represent output from TRAP where “initial” represents the input value required 

as a first guess to generate a model and where “final” represents actual statistic 

given by the fitted model equation.

L og(TAN mg/L )

Surviving Snai ls

-1.0 -.5 0 .5 1.0 1.5 2.0

0

.5

1.0

1.5

2.0

2.5

3.0

3.5

Initial Final Value Std. Error 95% LCL 95% UCL

X50 1 0.98487 6.01E-02 0.8489 1.1208

Slope 1.50E+00 1.56E+00 4.64E-01 5.11E-01 2.6093

Yint 3 2.9413 0.19206 2.5068 3.3758

Effect Level EC(X) 95% LCL 95% UCL

50 9.6576 7.0616 13.208

20 5.791 3.4781 9.642

10 4.2936 2.1723 8.4865

5 3.2591 1.3917 7.6325
22

Figure 2. Graphical display of the variability in measured water temperatures over the 28-

day test period amongst replicate chambers within the various test treatments 

(based on nominal TAN concentrations).

Control

Test Day

0 4 8 12 16 20 24 28

Temperature (°C)

21

22

23

24

25

26

27

0.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Temperature (°C)

21

22

23

24

25

26

27

1.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Temperature (°C)

21

22

23

24

25

26

27

3.8 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Temperature (°C)

21

22

23

24

25

26

27

7.5 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Temperature (°C)

21

22

23

24

25

26

27

15 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Temperature (°C)

21

22

23

24

25

26

27

Replicate 1

Replicate 2

Replicate 3

Replicate 4
23

Figure 3. Graphical display of the variability in measured pH of test water over the 28-day 

test period amongst replicate chambers within the various test treatments (based 

on nominal TAN concentrations)

Control

Test Day

0 4 8 12 16 20 24 28

pH

7.0

7.5

8.0

8.5

9.0

0.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

pH

7.0

7.5

8.0

8.5

9.0

1.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

pH

7.0

7.5

8.0

8.5

9.0

3.8 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

pH

7.0

7.5

8.0

8.5

9.0

7.5 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

pH

7.0

7.5

8.0

8.5

9.0

15 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

pH

7.0

7.5

8.0

8.5

9.0

Replicate 1

Replicate 2

Replicate 3

Replicate 4
24

Figure 4. Graphical display of the variability in measured dissolved oxygen concentrations 

over the 28-day test period amongst replicate chambers within the various test 

treatments (based on nominal TAN concentrations).

Control

Test Day

0 4 8 12 16 20 24 28

Dissolved Oxygen (mg/L)

0

2

4

6

8

10

0.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Dissolved Oxygen (mg/L)

0

2

4

6

8

10

1.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Dissolved Oxygen (mg/L)

0

2

4

6

8

10

3.8 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Dissolved Oxygen (mg/L)

0

2

4

6

8

10

7.5 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Dissolved Oxygen (mg/L)

0

2

4

6

8

10

15 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Dissolved Oxygen (mg/L)

0

2

4

6

8

10

Replicate 1

Replicate 2

Replicate 3

Replicate 4
25

Figure 5. Graphical display of the variability in measured TAN concentrations over the 28-

day test period amongst replicate chambers within the various test treatments 

(based on nominal TAN concentrations). Line represents the daily average TAN 

from all replicates within a treatment when measured, and interpolation between 

the daily average concentrations when measurements were not made.

Control

Test Day

0 4 8 12 16 20 24 28

Total Ammonia Nitrogen (mg TAN/L) 0.0

0.2

0.4

0.6

0.8

1.0

0.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Total Ammonia Nitrogen (mg TAN/L) 0.0

0.3

0.6

0.9

1.2

1.5

1.8

1.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Total Ammonia Nitrogen (mg TAN/L) 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.8 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Total Ammonia Nitrogen (mg TAN/L) 0

1

2

3

4

5

7.5 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Total Ammonia Nitrogen (mg TAN/L) 0

1

2

3

4

5

6

7

8

15 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Total Ammonia Nitrogen (mg TAN/L) 8

10

12

14

16

18

20

Replicate 1

Replicate 2

Replicate 3

Replicate 4

Daily Average
26

Figure 6. Graphical display of the variability in calculated unionized ammonia (UIA) 

concentrations over the 28-day test period amongst replicate chambers within the 

various test treatments (based on nominal TAN concentrations). Line represents 

the daily average UIA from all replicates within a treatment when measured, and 

interpolation between the daily average concentrations when measurements were 

not made.

Control

Test Day

0 4 8 12 16 20 24 28

Unionized Ammonia (mg UIA/L) 0.000

0.005

0.010

0.015

0.020

0.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Unionized Ammonia (mg UIA/L) 0.00

0.05

0.10

0.15

1.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Unionized Ammonia (mg UIA/L) 0.00

0.05

0.10

0.15

0.20

0.25

0.30

3.8 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Unionized Ammonia (mg UIA/L) 0.0

0.2

0.4

0.6

0.8

7.5 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Unionized Ammonia (mg UIA/L) 0.0

0.3

0.6

0.9

1.2

1.5

15 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

Unionized Ammonia (mg UIA/L) 0.0

0.5

1.0

1.5

2.0

Replicate 1

Replicate 2

Replicate 3

Replicate 4

Daily Average
27

Figure 7. Twenty eight day survival of P. canaliculata across an ammonia concentration gradient with replicate values 

averaged. P. canaliculata in the control treatment were maintained for an additional 14 days after test completion to 

further assess controls.

0

2 0

4 0

6 0

8 0

100

120

0 2 3 4 5 7 9 1 2 1 4 1 6 1 9 2 1 2 3 2 6 2 8 3 3 3 5 3 6 3 7 3 8 3 9 4 0 4 1 4 2

Survival (%)

Test Day

Control

0.34 mg TAN/L

0.81 mg TAN/L

1.86 mg TAN/L

4.50 mg TAN/L

11.98 mg TAN/L
28

Figure 8. Twenty-eight day survival of P. canaliculata across an ammonia concentration 

gradient by replicate chamber. Note the anomalous early mortality in replicates 3 

and 4 of the nominal 1.9 mg TAN/L treatment.

Control

Test Day

0 4 8 12 16 20 24 28

# Live Snails

0

2

4

6

0.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

# Live Snails

0

2

4

6

1.9 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

# Live Snails

0

2

4

6

3.8 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

# Live Snails

0

2

4

6

7.5 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

# Live Snails

0

2

4

6

15 mg TAN/L Nominal Concentration

Test Day

0 4 8 12 16 20 24 28

# Live Snails

0

2

4

6

Replicate 1

Replicate 2

Replicate 3

Replicate 4
29

Figure 9. Graphical display of concentration-response results for P. canaliculata after 28 

days in response to TAN, (a) all replicates from all treatments included, and (b) 

minus the four replicates from the second lowest (nominal 1.9 mg TAN/L) 

treatment. TRAP logistic equation results are as those presented in Table 5.

28 Day P. canaliculata Survival

(All Treatments)

log (mg TAN / L)

-1.0 -0.5 0.0 0.5 1.0

Proportion of Live Snails

0.0

0.2

0.4

0.6

0.8

1.0

Replicate 1

Replicate 2

Replicate 3

Replicate 4

28 Day P. canaliculata Survival

(1.9 mg TAN/L Nominal Concentration Excluded)

log (mg TAN / L)

-1.0 -0.5 0.0 0.5 1.0

Proportion of Live Snails

0.0

0.2

0.4

0.6

0.8

1.0
30

APPENDIX 1. Justification for selection of test species and species distribution.

GLEC’s test species selection process utilized a “tiered” approach to identify possible candidates 

with the widest distribution range in the continental United States and with habitat that would 

most likely receive discharge from municipal wastewater treatment plants, i.e., both small and

large rivers. Step one in the screening process was to identify families of snail species that were 

non-pulmonates (gill-bearing species; formerly referred to as prosobranchs). A USEPA 

document: USEPA. 1982. Freshwater snails (mollusca: gatropoda) of North America. EPA600/3-82-026, was used as the primary tool for identifying ten families of non-pulmonates in 

North America representing 49 genera and 349 species. This identification guide was used to 

eliminate those families which were amphibious breathers (Ampullariidae), were not freshwater 

species (Neritinidae), had highly-specialized habitats requirements (Micromelaniidae, cavedwelling) or were not widely distributed (Pomatiopsidae and Thiaridae). From the initial list of 

ten families, the EPA document was utilized a third time to identify the genera within those 

remaining families that had large species distributions. As a result of this third tier screen, 20 

genera were identified as worthy to pursue given the requirements of the project.

The 20 genera identified were entered into the NatureServe Explorer website 

(http://www.natureserve.org/explorer/servlet/NatureServe?init=Species). NatureServe Explorer 

is a source for information of over 70,000 plants and animals of the United States and Canada. 

When the genera were entered, only the top three species within each genus in terms of the 

greatest geographical distribution (most states and/or area) were noted to create a list of possible 

test species. This list did not include any invasive species or species that were listed as critically 

endangered or threatened. From this fourth screen 10-15 species were identified that could be 

used for ammonia toxicity testing.

GLEC staff then contacted experts who could help in the process of choosing from the list the 

best test species with known distributions in large rivers that were not highly specialized, and 

were readily available for collection and subsequent testing. Dr. Paul Johnson, from the 

Alabama Aquatic Biodiversity Center was fundamental in identifying species from the list that 

were relatively ubiquitous and were expected in large rivers. He also provided assistance with 

his extensive snail culturing experience to help eliminate those species that would prove too 

difficult to handle for testing. Dr. Thomas Watters from the Ohio State University’s Division of 

Mollusks in the Department of Evolution, Ecology and Organismal Biology provided assistance 

with identifying species that were present in Ohio and available for collection. Dr. Watters also 

provided GLEC staff with contact information for additional experts who knew of specific 

locations of candidate species. In addition, he allowed us to observe and identify juvenile 

specimens in his culture tank of Pleurocera canaliculata, providing ease of identification in the 

field. Based on Dr. Watter’s and Johnson’s recommendations, three species of snails were 

identified for testing. Patricia Morrison of the U.S. Fish and Wildlife Service, Ohio River 

Islands National Wildlife Refuge provided assistance in identify locations along the Ohio River 

where two of the three species (Birgella subglobosus and P. canaliculata and) could be collected 

for testing. The third species Campeloma decisum is also known to inhabit large rivers and can 

be found in large numbers in the Cuyahoga River, according to Dr. Martin Huehner, 

EnviroScience, Inc. Another benefit of the three test species selected is that they represent three 

of the most widely-distributed families of non-pulmonate snail species in the U.S. B. 
31

subglobosus, P. canaliculata, and C. decisum are from the families Hydrobiidae, Pleuroceridae, 

and Viviparidae, respectively.

Maps showing the distribution of the selected snail test species (depicted as a pattern dots) 

overlaid on a base layer that is a gradient scale of the number of snail families (non-pulmonates) 

present in each state are provided for reference (attached separately below in this Appendix for 

convenience). All data are from NatureServe Explorer.
32

Map 1. Distribution of globe siltsnail (B. subglobosus) within the conterminous U.S.
33

Map 2. Distribution of silty hornsnail (P. canaliculata) within the conterminous U.S.
34

Map 3. Distribution of pointed campeloma (C. decisum) within the conterminous U.S.
35

APPENDIX 2. Summary of acquisition, acclimation, maintenance, and health of test snail 

species.

Birgella subglobosus (globe siltsnail)

General Species Information:

The genus Birgella currently contains a single widespread species (Freshwater Mollusk 

Conservation Society, 2004) that has a wide distribution across the United States (see Map 1, 

Appendix 1). It has been recorded from Alabama, Arkansas, Iowa, Kentucky, Michigan, 

Missouri, and New York, and is found in large streams, rivers and lakes. 

The globe siltsnail is part of the larger Family Hydrobiidae which is composed of over 500 

genera and several thousand species (largely freshwater) worldwide. The fauna of the United 

States and Canada from this large taxonomic group is among the most diverse of the world, with 

49 genera and 500 plus species (350 currently named). The most widespread genera are 

Amnicola, Birgella, Cincinnatia, Probythinella, Pyrgulopsis, Somatogyrus and Tryonia. The 

largest genus is Pyrgulopsis with over 140 known species, the vast majority of which are 

described as being restricted to springs and small streams of the arid western areas of the country 

(Freshwater Mollusk Conservation Society, 2004).

The shell morphology varies widely from flattened planispiral shaped to conic to elongated. The 

size varies from about a 1 mm to almost 10 mm in height. Anatomically the group is very diverse 

with most generic and species designations primarily determined by anatomical characters, 

particularly penial morphology. The group feeds on detritus and bacterial/algal films and can 

inhabit slow to fast moving water. All the North American taxa have separate sexes and lay 

single egg capsules, however the introduced species, Potamopyrgus antipodarum is 

parthenogenetic, which partially accounts for its success as an invasive species (Freshwater 

Mollusk Conservation Society, 2004).

Acquisition:

Approximately 300 mature snails were collected August 11, 2010 along the Ohio River at 

Belleville, Wood County, West Virginia (Latitude: 39o08’21.03”; Longitude: 81o44’25.63”, 

elevation 595 feet). Snails were collected by hand using mask and snorkel. Snails were bagged 

(approximately 50 per bag) in site water super-saturated with pure oxygen, and subsequently 

shipped (styrofoam insulated boxes cooled with ice) overnight to the laboratory in Traverse City, 

Michigan. Temperature of Ohio River water upon collection was approximately 24oC. Water 

quality conditions of the bagged site water (with snails) measured upon receipt at the laboratory 

were:

Specific conductivity – 501 μmhos/cm

Dissolved oxygen – 20 mg/L

pH – 7.25

Water temperature – 23.4oC

Alkalinity – 74 mg CaCO3/L

Hardness – 168 mg CaCO3/L

Holding and Acclimation Process:

Upon receipt at the laboratory, snails were immediately transferred to a dedicated on-site 
36

holding/test facility and released into a 20 gallon tank filled with the Ohio River water (gently 

aerated) in which they were shipped. The snails were initially allowed to feed ad libitum only on 

the periphyton colonizing the sides of the holding tank, after which (beginning August 17, 2010) 

they were fed nutrients and PVC tiles covered with periphyton from in-house fathead minnow 

culture tanks. 

One day after arrival at the laboratory (on August 13, 2010), approximately 25 percent of the 

Ohio River water was replaced with Lake Michigan acclimation/test dilution water. Snails were 

maintained for two days under these conditions, and on August 16, 2010, another 25 percent of 

holding water was replaced with acclimation/test dilution water (Lake Michigan water) resulting 

in the following conditions:

Specific conductivity – 468 μmhos/cm

Dissolved oxygen – 7.7 mg/L

pH – 7.80

Water temperature – 24.1oC

Alkalinity – approximately 124 mg CaCO3/L

Hardness – 168 mg CaCO3/L 

Five days after receipt at the laboratory (on August 17, 2010), and periodically thereafter, the 

snails received supplemental nutrients via the addition of a natural organic matter slurry mix 

from an existing in-house fathead minnow culture fed frozen brine shrimp. Ammonia measured 

in holding water was not detectable (<0.06 mg TAN/L) throughout acclimation and holding.

One week after receipt (August 18, 2010), the snail holding tank began receiving flowing Lake 

Michigan test dilution water at 160 mls/min. 

Maintenance and Health:

Snails were allowed to graze on algae-laden tiles collected from the in-house fathead minnow 

culture tanks ad libitum, and periodically received supplemental nutrients (natural organic matter 

slurry mix) as described above. Water quality during holding consisted of the following ranges:

DO – 7.4 to 7.8 mg/L

pH – 8.13- 8.42

Water temperature – 23.2 to25.1oC

Approximately 2 weeks after receipt (August 26, 2010), 130 dead snails were removed from the 

tank leaving only 167 survivors. Excessive mortality continued through August 30, 2010 when 

plans for testing this particular snail species were aborted after further consultation with EPA.

Other Information:

None
37

Pleurocera canaliculata (silty hornsnail)

General Species Information:

The silty hornsnail (Family Pleuroceridae) is also relatively widely distributed across the United 

States (see Map 2, Appendix 1). It has been recorded in the Mississippi River and Great Lakes 

drainages (including into Ontario and British Columbia, Canada), in the Hudson River basin, and 

in the Gulf of Mexico drainages. It prefers slow current areas of streams and rivers near the bank 

where it often leaves trails in the substrate (Freshwater Mollusk Conservation Society, 2004).

The shells of this species are narrowly to elongately conic or cylindrical. Aperture is rectangular, 

with a short anterior canal, 20-40 mm in height. Shell sculpture varies from smooth to with spiral 

ribs, lirates or malleations and occasionally short blunt spines.

Pleurocerids in general are all dioecious (separate sexes), the males lack penises, and the females 

possess an egg-laying pore or groove on the right side of the foot. Little is currently known about 

the ecology of this family of snails. Snails of the genus Pleurocera prefer the silty, low-flow 

areas along the bank of streams, while Elimia and Leptoxis are commonly found in fast-moving 

waters in the mid-channel, attached to or underneath rocks. Pleurocerids are primarily algal 

grazers. They often occur in large numbers, frequently far outnumbering species of other 

gastropod families in freshwater streams. Elimia can be particularly abundant in some locales, 

often numbering hundreds per square meter. Pleurocerid adults have thick shells, making them 

less susceptible to predation than thinner-shelled gastropods like physids (Freshwater Mollusk 

Conservation Society, 2004).

Acquisition:

Approximately 280 snails (238 large and 46 smaller snails) were collected August 11, 2010 

along the Ohio River at Belleville, Wood County, West Virginia (Latitude: 39o08’21.03”; 

Longitude: 81o44’25.63”, elevation 595 feet). Snails were collected by hand using mask and 

snorkel. Snails were bagged (approximately 50 per bag) in site water super-saturated with pure 

oxygen, and subsequently shipped (styrofoam insulated boxes cooled with ice) overnight to the 

laboratory in Traverse City, Michigan. Temperature of Ohio River water upon collection was 

approximately 24oC. Water quality conditions of the bagged site water (with snails) measured 

upon receipt at the laboratory were:

Specific conductivity – 504 μmhos/cm

Dissolved oxygen – 13.4 mg/L

pH – 7.09

Water temperature – 24.3oC

Alkalinity – 74 mg CaCO3/L

Hardness – 176 mg CaCO3/L

Holding and Acclimation Process:

Upon receipt at the laboratory (August 12, 2010), all snails were immediately transferred to a 

dedicated on-site holding/test facility and released into a 20 gallon tank (large snails) or 500 ml 

beaker (small snails) filled with the Ohio River water (gently aerated) in which they were 

shipped. As above, all snails were initially allowed to feed ad libitum only on the periphyton 

colonizing the sides of the holding tank or beaker, after which (beginning August 17, 2010) they 

were fed PVC tiles covered with periphyton from in-house fathead minnow culture tanks. Note: 
38

as many as 40 large snails were placed (upon arrival) in a separate large beaker and fed an algae 

and commercial fish flake food slurry as a small feeding trial. 

One day after receipt at the laboratory (August 13, 2010), approximately 25 percent of the Ohio 

River water was replaced with Lake Michigan acclimation/test dilution water. Snails were 

maintained two days under these conditions, and on August 16, 2010, another 25 percent of 

holding water was replaced with acclimation/test dilution water resulting in the following 

conditions:

Specific conductivity – 471 μmhos/cm

Dissolved oxygen – 6.2 mg/L

pH – 7.29

Water temperature – 24.2oC

Alkalinity – approximately 124 mg CaCO3/L

Hardness – 156 mg CaCO3/L

Five days after receipt at the laboratory (August 17, 2010) and periodically thereafter the snails 

received supplemental nutrients via the addition of a natural organic matter slurry mix from an 

existing in-house fathead minnow culture and PVC tiles coated with periphyton. Ammonia 

measured in holding water was not detectable (<0.06 mg TAN/L) throughout acclimation and 

holding.

One week after receipt (August 18th, 2010), the snail holding tank began receiving flowing Lake 

Michigan test dilution water at 160 mls/min (after 25% of water was replaced with L MI water 3 

times). 

Maintenance and Health:

Snails were allowed to graze on algae-laden tiles collected from the in-house fathead minnow 

culture tanks ad libitum, and periodically received supplemental nutrients (natural organic matter 

slurry mix) as described above. Water quality during holding consisted of the following ranges:

DO – 7.4 to 8.3 mg/L

pH – 8.13- 8.37

Water temperature – 23.3 to 26.2oC

By August 26, 2010 (approximately 2 weeks after receipt), there were a total of 236 large and 45 

smaller (presumed juvenile) snails remaining in the holding tank (<0.1 mortality). By September 

14, 2010 (prior to initiation of the range-finding test with this species), there were 205 large and 

45 small snails remaining. Survival of large snails immediately prior to 28-day toxicity testing

(or after approximately 6 weeks holding) was 80 percent (98 percent for small snails).

Other Information:

A 96-hr range-finding test was initiated on September 14, 2010 with large and small P. 

canaliculata. The range-finding test included a sample size of three snails tested in duplicate 

chambers (N= 6 snails total of each size) at each of five concentrations with a control. The 

average measured ammonia concentrations (N=3) for the test were 2.6, 3.6, 7.6, 17.0, and 32.8 

mg TAN/L, respectively. The resulting 96-hour EC50 was 9.1 mg TAN/L with 95 percent 

confidence intervals ranging from 6.0 to 15.0 mg TAN/L for the large P. canaliculata (see Table 

2 of this report), and 7.9 mg TAN/L with 95 percent confidence intervals ranging from 4.8 to 

13.4 mg TAN/L for the small snails. The average test pH was 8.18. The average test temperature 
39

was 24.8oC. 

From these preliminary results, it appears that smaller, possibly juvenile, P. canaliculata are at 

least as acutely sensitive to ammonia as larger, presumably more mature snails.
40

Campeloma decisum (pointed campeloma)

General Species Information:

Combined with all its probable synonyms, Campeloma decisum is widespread throughout eastern 

North America, ranging from well into Canada and down to Florida (see Map 3, Appendix 1). 

This snail species prefers the sandy bottoms of rivers where the current is sufficient to oxygenate 

the water column (Freshwater Gastropods of North America 

at:http://spinner.cofc.edu/~fwgna/species/viviparidae/c_decisum.html?referrer=webcluster&, 

accessed January 2011).

The burrowing habit and peculiar radular morphology displayed by these snails imply an ability 

to filter feed, as has been documented for other snails in the Family Viviparidae, but the snails 

are assumed to have retained the ability to graze or harvest deposits on soft sediments as well. 

This species has also been observed feeding directly on carrion (Dr. Martin Huehner, 

EnviroScience, Inc., personal communication).

Campeloma are ovoviviparous brooders where parthenogenesis has evolved. Some populations 

of Campeloma appear comprised entirely of parthenogenic females, others appear to reproduce 

entirely by outcrossing, and some populations display a mixture of the two modes. Only one 

year is required for maturation in some populations, two in others, and both semelparous and 

iteroparous reproduction have been reported (see references associated with weblink provided 

above).

Acquisition:

Approximately 400 snails were collected from the Upper Cuyahoga River via subcontract with 

Dr. Martin Huehner of EnviroScience, Inc., on September 28, 2010. The undisclosed collection 

site has very clean water that arises from a large watershed managed by the City of Akron for 

drinking water. Snails were collected via scuba diving by sieving sediments. Captured animals 

were maintained overnight in clean plastic buckets filled with continually aerated ambient river 

water before delivery to Great Lakes Environmental Center in Columbus, Ohio for overnight 

shipping to their laboratory in Traverse City, Michigan, as described above. Water quality 

conditions of the bagged site water (with snails) measured upon receipt at the laboratory were:

Dissolved oxygen – 11 mg/L

Water temperature – 16.4 to 17.8oC

Holding and Acclimation Process:

On September 30, 2010, snails were transferred to the dedicated holding/test facility and released 

(N = 201 and 196 snails, respectively) into two 20 gallon tanks filled with the Upper Cuyahoga 

River water (gently aerated) in which they were shipped. The bottoms of the tanks were covered 

with approximately 1 inch of inert sand for burrowing and cover. Snails were fed a natural 

organic matter slurry mix from in-house fish culture tanks as described above.

On October 1, 2010, approximately 25 percent of the upper Cuyahoga River water was replaced 

with Lake Michigan acclimation/test dilution water. Holding tank water temperature was 23oC. 

The same regimen was implemented over the course of next two to four days until approximately 

one week after receipt, the snail holding tanks began receiving flowing Lake Michigan test 

dilution water at approximately 160 mls/min. 
41

Maintenance and Health:

Snails were fed the natural organic matter slurry mix two to three times per week for the first two 

weeks. Survival was 96 percent until October 15, 2010, declining progressively thereafter to 69 

percent by October 28th. A one-inch layer of natural Boardman River organic sediment was 

added to the inert sandy layer at this time to improve foraging and cover.

At this time, based upon consultation with an outside expert, diet was switched to a commercial 

spirulina-based flake fish food, and, with EPA approval, GLEC initiated a 1°C per day 

temperature reduction in holding tanks from 25°C to 20°C – the new target water temperature for 

the pending 28-day toxicity test with the species. 

Under the new holding and feeding conditions, mortality was reduced to just a 20 percent loss of 

animals from November 8th until December 2nd, 2010, when a 96-hr range-finding test was 

initiated (see below).

Plans for testing this particular snail species were subsequently aborted after further consultation 

with EPA based partially on only 67 percent survival of Campeloma by December 7, 2010.

Other Information:

A 96-hr range-finding test was initiated on December 2, 2010 with the snail species. From these 

preliminary results, it appears that this snail species (C. decisum) is at least as acutely sensitive to 

ammonia as P. canaliculata.
42

APPENDIX 3. Characterization of dilution water from East Arm Grand Traverse Bay, 

Lake Michigana.

Parameter Level of Detection (mg/L)

2/12/2010 

Result (mg/L)

2/16/2010 

Result (mg/L)

Total Aluminum 0.05 ND 0.05

Dissolved Aluminum 0.05 ND ND

Total Antimony 0.002 ND ND

Total Arsenic 0.002 0.003 ND

Total Barium 0.05 ND ND

Total Beryllium 0.001 ND ND

Total Cadmium 0.001 ND ND

Total Calcium 0.1 42.6 32.8

Total Chromium 0.005 ND ND

Total Cobalt 0.02 ND ND

Total Copper 0.004 ND ND

Total Iron 0.05 ND ND

Total Lead 0.002 ND ND

Total Magnesium 5.0 13.7 12.3

Total Manganese 0.05 ND ND

Total Mercury 0.0005 ND ND

Total Nickel 0.05 ND ND

Total Potassium 0.1 1.42 1.33

Total Selenium 0.005 0.006 ND

Total Silver 0.001 ND ND

Total Sodium 0.5 8.83 7.75

Total Thallium 0.002 ND ND

Total Vanadium 0.004 ND ND

Total Zinc 0.05 ND ND

a

Analysis of City Tapwater (from adjacent East Arm Grand Traverse Bay- 02/11) indicated no detects for all carbamates, 

chlorinated acid herbicides and pesticides, and very low levels of only a few volatile organic compounds. Chloride and 

sulfate concentrations were 7 and 20 mg/L, respectively, and nitrate and nitrite (as N) were below reporting limits of 0.4 

and 0.05 mg/L, respectively.

ND = Below level of detection.
43

APPENDIX 4. Cross validation of archived samples using new standard curve.

(ppm)

0.1 -2.302585093 30.5

0.5 -0.693147181 11.3

1 0 -6.8

10 2.302585093 -63.2

20 2.995732274 -81

SUMMARY OUTPUT

Regression Statistics

Multiple R 0.990922238

R Square 0.981926881

Adjusted R Square 0.975902508

Standard Error 7.474824291

Observations 5

ANOVA

df S S MS F Significance F

Regression 1 9106.873005 9106.873 162.992381 0.001036835

Residual 3 167.6189945 55.872998

Total 4 9274.492

Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0%

Intercept -11.75738543 3.434865987 -3.422953 0.04175261 -22.688662 -0.826109 -22.688662 -0.82610886

X Variable 1 -21.89411936 1.714919858 -12.76685 0.001036835 -27.3517597 -16.43648 -27.35176 -16.436479

CALIBRATION

CONCENTRATIONS Natural Log Millivolts

NA na NA Blank 75 -3.962588 0.019013833 0.000

P.canaliculata10/20/10 Con 1 8700 43.9 -2.542116 0.078699715 0.05969 J 0.0597

P.canaliculata10/12/10 A 1 8364 -0.6 -0.509606 0.600731923 0.58172 0.582

P.canaliculata10/12/10 A 3 8366 -1.2 -0.482202 0.617422406 0.59841 0.598

P.canaliculata10/19/10 A 3 8690 1.5 -0.605523 0.545789089 0.52678 0.527

P.canaliculata10/14/10 A 4 8643 -0.1 -0.532444 0.587168365 0.56815 0.568

P.canaliculata10/15/10 B 1 8660 -20.1 0.3810436 1.46381146 1.44480 1.445

P.canaliculata10/12/10 B 2 8369 -10.8 -0.043728 0.957214312 0.93820 0.938

P.canaliculata10/12/10 C 4 8372 -25.3 0.6185503 1.856235147 1.83722 1.837

P.canaliculata10/20/10 C 4 8713 -32 0.9245686 2.520780479 2.50177 2.502

P.canaliculata10/12/10 D 2 8373 -47.5 1.6325212 5.116758974 5.09775 5.098

Reporting 

Ammonia

Level

Test 

Organism

Sample 

Date Replicate

Sample 

Bottle ID Millivolts

Natural 

Log (conc)

Predicted 

Ammonia 

Levels

Ammonia 

Minus Blank

KEY:

MDL = 0.04

RL = Reporting Limit (lowest standard, 0.1)

J = Between MDL and RL

U = Below MDL (non detect)

NA = Not Applicable
44

APPENDIX 5A. Water quality measurements Treatment A (control).

Temp pH DO SC Temp pH DO SC Temp pH DO SC Temp pH DO SC

9/22/10 0 25.5 8.02 7.4 274 25.5 8.07 7.4 315

9/23/10 1 25.6 25.0 7.86 7.0 7.90 6.7

9/24/10 2 26.0 8.14 6.9 302 25.7 8.17 7.1 312 25.9 8.14 7.0 315 26.1 8.33 7.2 320

9/25/10 3 24.2 8.07 7.1 24.4 8.12 7.0 24.0 8.14 7.1 24.4 8.20 7.2

9/26/10 4 24.2 7.96 7.9 24.3 8.11 7.5 23.3 8.13 7.5 24.3 8.20 7.9

9/27/10 5 23.9 8.12 8.0 324 24.0 8.18 8.1 324 23.9 8.19 7.6 319 24.3 8.18 7.8 312

9/28/10 6 26.0 8.12 7.6 25.6 8.21 7.7 25.4 8.27 7.5 26.1 8.31 8.0

9/29/10 7 24.6 8.01 7.5 24.8 8.05 7.6 24.4 8.09 7.4 24.8 8.12 7.6

9/30/10 8 25.7 8.09 7.6 25.6 8.19 7.6

10/1/10 9 23.4 7.96 7.4 23.3 7.94 5.6

10/2/10 1 0 22.3 7.99 7.7 21.7 F 8.02 7.8

10/3/10 1 1 22.6 8.43 8.1 22.5 8.28 8.2

10/4/10 1 2 24.4 8.07 7.1 311 24.3 7.86 6.6 311

10/5/10 1 3 24.3 8.22 7.5 24.6 8.25 7.6 24.8 8.44 7.5 25.1 8.43 7.6

10/6/10 1 4 24.3 8.02 7.8 24.5 8.25 7.8 24.4 8.36 7.4 24.6 8.39 8.0

10/7/10 1 5 25.7 8.13 7.5 25.7 8.32 8.0 25.5 8.26 7.5 25.8 8.33 7.8

10/8/10 1 6 24.4 8.31 7.3 24.5 8.30 7.4 24.6 8.25 7.6 24.5 8.28 7.6

10/9/10 1 7 25.3 8.25 7.6 25.2 8.30 7.7 25.1 8.21 7.3 24.5 8.30 7.5

10/10/10 1 8 24.7 8.25 7.4 24.6 8.30 7.4 24.3 8.28 7.2 24.7 8.33 7.7

10/11/10 1 9 25.4 8.22 7.8 308 25.5 8.20 7.7 310 25.3 8.12 7.8 304 24.6 8.10 7.7 299

10/12/10 2 0 24.7 8.01 7.7 24.5 8.16 7.9 24.3 8.16 7.7 24.4 8.26 8.1

10/13/10 2 1 25.1 8.20 7.7 25.0 8.32 7.7 24.9 8.16 7.4 25.3 8.12 7.4

10/14/10 2 2 25.6 8.20 7.8 25.5 8.30 8.1 25.2 8.25 7.6 25.7 8.29 8.0

10/15/10 2 3 25.4 8.70 8.1 25.4 8.73 8.3 25.0 8.68 7.9 25.7 8.65 8.3

10/16/10 2 4 24.6 8.23 8.0 24.6 8.23 8.2 24.1 8.19 7.8 24.8 8.23 8.0

10/17/10 2 5 25.5 8.31 8.0 25.5 8.30 8.0 25.4 8.29 7.9 25.8 8.26 8.0

10/18/10 2 6 24.8 8.21 8.7 294 24.7 8.29 9.0 301 24.3 8.22 8.3 304 25.0 8.29 8.5 306

10/19/10 2 7 24.5 8.36 8.5 24.0 8.45 8.7 24.2 8.21 8.4 24.6 8.39 8.2

10/20/10 2 8 24.5 8.06 8.2 291 24.5 8.36 8.5 301 24.5 8.31 8.5 304 24.6 8.35 8.3 308

DATE

TEST 

DAY

Replicate 1 Replicate 2 Replicate 3 Replicate 4
45

APPENDIX 5B. Water quality measurements Treatment B (nominal 0.9 mg TAN/L).

Temp pH DO SC Temp pH DO SC Temp pH DO SC Temp pH DO SC

9/22/10 0 25.9 8.13 7.3 339 26.0 8.17 7.4 340

9/23/10 1 25.7 25.5 7.85 7.0 7.98 7.0

9/24/10 2 25.7 8.01 5.9 330 25.9 7.79 5.5 336 25.9 8.06 5.2 317 26.0 8.16 7.3 321

9/25/10 3 24.0 8.11 6.7 24.3 8.11 6.8 24.2 8.13 7.0 24.3 8.12 7.1

9/26/10 4 23.6 8.08 6.7 24.0 7.99 6.2 24.0 8.15 7.9 24.1 8.20 8.0

9/27/10 5 23.6 8.20 7.8 326 24.2 8.20 8.1 328 23.7 8.19 8.0 329 23.9 8.20 7.9 329

9/28/10 6 25.4 8.35 7.8 26.1 8.35 7.8 25.5 8.30 7.5 24.9 8.21 6.7

9/29/10 7 24.7 8.15 7.3 24.9 8.10 7.5 24.6 8.02 6.9 23.4 8.05 7.1

9/30/10 8 25.5 8.22 7.6 25.5 8.18 7.6

10/1/10 9 23.2 7.93 7.2 23.5 7.43 5.6

10/2/10 1 0 22.0 7.57 3.0 F 21.2 F 7.64 5.2

10/3/10 1 1 22.6 8.58 8.0 22.6 7.50 2.1 F

10/4/10 1 2 24.4 8.24 7.2 322 24.5 7.86 6.5 319

10/5/10 1 3 24.1 8.25 7.6 24.8 8.21 7.5 25.0 8.41 7.8 24.7 8.62 7.6

10/6/10 1 4 24.0 8.23 7.7 24.4 8.29 7.8 24.6 8.32 7.8 24.2 8.32 7.6

10/7/10 1 5 25.3 8.29 7.5 25.2 8.15 7.6 26.0 8.30 6.9 25.2 8.30 7.5

10/8/10 1 6 24.1 8.09 7.3 24.1 7.84 7.4 24.6 8.22 7.7 24.1 8.06 6.7

10/9/10 1 7 24.5 8.26 7.3 24.5 8.03 6.3 24.5 8.22 7.4 25.1 8.15 7.3

10/10/10 1 8 24.3 8.25 7.5 24.0 8.03 6.1 24.8 8.28 7.8 24.4 8.33 7.6

10/11/10 1 9 25.3 8.15 7.2 320 25.2 7.88 6.0 323 25.7 8.12 7.3 320 25.3 8.14 7.0 317

10/12/10 2 0 24.2 8.20 8.0 24.1 8.10 7.0 24.8 8.25 7.8 24.3 8.25 8.0

10/13/10 2 1 24.9 8.24 7.6 24.8 7.92 6.2 25.0 8.23 7.5 25.0 8.17 7.5

10/14/10 2 2 25.5 8.28 7.9 25.3 8.10 7.0 25.7 8.16 7.7 25.7 8.20 7.7

10/15/10 2 3 25.4 8.56 8.2 25.3 8.70 8.1 25.2 8.68 8.0 25.0 8.30 8.2

10/16/10 2 4 24.0 8.22 7.9 24.4 8.20 8.0 24.6 8.21 7.9 24.6 8.14 7.8

10/17/10 2 5 25.3 8.26 7.7 25.8 8.26 7.7 25.6 8.27 7.7 25.6 8.26 7.5

10/18/10 2 6 24.4 8.27 8.9 311 24.8 8.23 8.4 314 24.8 8.23 8.6 314 24.8 8.23 8.6 314

10/19/10 2 7 24.3 8.30 8.1 24.6 8.18 8.2 24.2 8.30 8.5 24.7 8.20 8.2

10/20/10 2 8 24.6 8.32 8.2 315 24.7 8.21 8.1 318 24.6 8.26 8.0 317 24.4 8.04 7.9 318

DATE TEST DAY

Replicate 1 Replicate 2 Replicate 3 Replicate 4
46

APPENDIX 5C. Water quality measurements Treatment c (nominal 1.9 mg TAN/L).

Temp pH DO SC Temp pH DO SC Temp pH DO SC Temp pH DO SC

9/22/10 0 26.0 8.13 7.3 352 25.9 8.10 6.8 354

9/23/10 1 25.6 25.3 7.96 6.5 7.93 6.7

9/24/10 2 25.9 8.20 6.9 328 25.8 7.85 5.3 343 25.5 7.88 5.5 347 25.5 7.91 5.4 346

9/25/10 3 24.8 8.18 7.1 24.0 7.73 4.5 24.0 7.80 4.8 23.7 7.98 6.1

9/26/10 4 24.3 8.20 7.8 23.2 7.80 5.7 23.0 7.81 5.5 23.8 8.07 7.4

9/27/10 5 23.5 8.20 7.6 333 23.8 8.05 6.6 338 23.9 8.15 7.5 334

9/28/10 6 25.2 8.22 7.4 25.9 8.23 7.3 25.7 8.36 7.4

9/29/10 7 24.1 8.10 7.3 24.4 8.08 7.4 24.5 8.10 7.6

9/30/10 8 25.3 8.10 7.1 25.3 8.04 6.8

10/1/10 9 23.3 7.79 6.4 23.2 7.90 7.1

10/2/10 1 0 22.7 7.52 3.3 F 21.3 F 7.98 7.7

10/3/10 1 1 22.6 8.49 7.8 22.5 7.65 3.4 F

10/4/10 1 2 24.3 7.58 4.2 339 24.3 7.98 7.3 319

10/5/10 1 3 24.9 8.26 7.7 23.8 8.10 6.2 24.3 8.58 7.8

10/6/10 1 4 24.0 8.21 7.1 23.6 8.08 6.6 24.8 8.41 7.9

10/7/10 1 5 25.2 8.18 6.8 25.0 8.23 7.2 26.2 8.34 7.8

10/8/10 1 6 24.3 8.13 7.1 23.7 7.92 6.0 25.0 8.23 7.7

10/9/10 1 7 25.0 8.12 7.4 25.1 8.10 6.9 25.4 8.25 7.4

10/10/10 1 8 24.4 8.19 7.5 24.3 8.09 7.0 24.7 8.26 7.6

10/11/10 1 9 25.4 8.09 6.9 325 25.3 7.96 6.8 325 25.7 8.16 7.3 324

10/12/10 2 0 25.4 8.30 7.9 25.8 8.20 7.8 24.8 8.20 8.0

10/13/10 2 1 25.0 8.23 7.4 24.9 8.20 7.5 24.9 8.16 7.3

10/14/10 2 2 25.6 8.19 7.8 24.9 8.19 7.7 24.8 8.12 7.2

10/15/10 2 3 25.3 8.62 8.2 24.6 8.64 8.0 24.6 8.59 7.7

10/16/10 2 4 24.9 8.20 8.1 24.0 8.05 7.9 24.0 8.07 7.3

10/17/10 2 5 25.7 8.28 8.5 25.4 8.41 8.1 25.2 8.09 6.9

10/18/10 2 6 25.0 8.22 8.6 320 24.3 8.54 10.1 318 24.0 8.04 7.1 311

10/19/10 2 7 24.4 8.33 8.5 24.3 8.41 8.3 24.0 8.19 7.5

10/20/10 2 8 24.9 8.25 8.4 324 24.2 8.32 8.2 313 8.52 8.5 315 24.0 8.16 7.2 324

DATE

TEST 

DAY

Replicate 1 Replicate 2 Replicate 3 Replicate 4
47

APPENDIX 5D. Water quality measurements Treatment D (nominal 3.8 mg TAN/L).

Temp pH DO SC Temp pH DO SC Temp pH DO SC Temp pH DO SC

9/22/10 0 25.9 8.16 7.4 361 25.9 8.13 7.1 363

9/23/10 1 25.3 25.2 7.89 6.9 25.7 7.85 6.7

9/24/10 2 25.7 7.84 5.1 354 26.1 8.18 6.8 338 26.0 7.71 4.2 358 23.7 7.76 5.2 362

9/25/10 3 23.8 7.80 4.9 24.6 8.13 7.0 24.6 8.16 7.1 23.1 7.87 5.0

9/26/10 4 23.3 7.82 5.6 24.3 8.17 7.8 24.3 8.20 7.7 23.0 7.80 5.3

9/27/10 5 23.2 7.92 6.0 354 23.6 8.20 7.8 346 24.2 8.20 7.9 317 25.1 7.93 5.5 349

9/28/10 6 25.1 8.13 6.2 25.5 8.29 7.5 26.3 8.30 7.7 23.7 7.97 5.5

9/29/10 7 24.0 7.89 5.8 24.3 8.08 7.3 25.1 8.12 7.7 7.87 5.4

9/30/10 8 25.2 7.92 5.6 25.2 8.08 6.8

10/1/10 9 23.5 7.27 2.9 F 23.3 8.03 7.1 21.8 F 7.49 1.9 F

10/2/10 1 0 22.3 8.12 8.0

10/3/10 1 1 22.8 8.28 6.6 22.6 8.23 8.2

10/4/10 1 2 24.8 8.08 6.1 343 24.8 7.82 6.0 333

10/5/10 1 3 24.9 8.30 7.8 25.5 8.27 7.7 24.2 8.47 6.5 23.8 8.34 6.5

10/6/10 1 4 24.6 8.30 7.9 25.0 8.24 7.9 23.6 8.13 6.9 23.4 8.11 7.2

10/7/10 1 5 25.6 8.12 7.9 26.0 8.30 7.8 25.4 8.30 7.0 24.9 8.12 7.0

1 6 24.8 8.12 7.1 24.3 8.27 7.8 24.0 7.94 6.3 24.0 7.93 6.0

10/9/10 1 7 25.3 8.16 7.4 25.6 8.27 7.5 25.2 8.25 7.5 24.6 8.03 7.0

10/10/10 1 8 24.7 8.21 7.4 25.0 8.26 7.6 24.0 8.10 6.9 24.2 8.00 6.0

10/11/10 1 9 25.6 8.11 7.9 339 25.7 8.20 8.1 338 26.0 8.23 7.4 338 25.3 8.02 6.5 341

10/12/10 2 0 24.6 8.26 7.7 24.9 8.42 7.9 25.0 8.09 6.7

10/13/10 2 1 24.9 8.23 7.5 25.4 8.24 7.6 24.5 8.29 8.2

10/14/10 2 2 25.7 8.16 7.9 25.7 8.20 7.9 24.9 8.02 6.9

10/15/10 2 3 25.6 8.57 8.2 25.7 8.63 8.2 24.7 8.29 6.8

10/16/10 2 4 24.8 8.22 8.1 24.7 8.23 7.7 24.3 8.10 8.2

10/17/10 2 5 25.7 8.28 7.8 25.8 8.20 7.8 25.7 8.09 7.2

10/18/10 2 6 24.8 8.28 8.5 332 24.6 8.27 8.6 331 24.9 8.25 8.6 335

10/19/10 2 7 24.4 8.34 8.5 24.6 8.35 8.5 24.7 8.29 8.4

10/20/10 2 8 24.4 8.34 8.0 333 24.6 8.31 8.1 335 24.6 8.32 8.2 331 24.7 8.30 8.1 336

DATE

TEST 

DAY

Replicate 1 Replicate 2 Replicate 3 Replicate 4
48

APPENDIX 5E. Water quality measurements Treatment E (nominal 7.5 mg TAN/L).

Temp pH DO SC Temp pH DO SC Temp pH DO SC Temp pH DO SC

9/22/10 0 25.8 8.11 7.2 383 25.7 8.09 7.3 387

9/23/10 1 25.2 25.1 7.96 6.7 8.00 6.7

9/24/10 2 26.0 8.08 6.4 360 26.2 8.08 6.6 361 26.2 8.12 6.6 361 26.3 8.02 6.4 362

9/25/10 3 23.7 8.05 6.8 24.0 8.06 6.7 24.4 8.09 6.9 24.6 8.04 6.4

9/26/10 4 23.6 8.10 7.6 23.7 8.17 7.8 23.8 8.14 7.6 24.0 8.12 6.9

9/27/10 5 23.9 8.10 7.3 367 23.8 8.14 7.3 337 24.1 8.17 7.5 376 23.8 8.15 7.5 368

9/28/10 6 25.4 8.19 7.5 25.4 8.23 7.1 25.4 8.23 7.5 26.2 8.28 7.5

9/29/10 7 24.1 8.05 7.6 24.3 8.09 7.3 24.5 8.08 7.5 24.9 8.11 7.6

9/30/10 8 25.1 8.07 7.3 25.2 8.13 7.4

10/1/10 9 23.3 7.86 6.6 23.3 7.80 6.0

10/2/10 1 0 22.1 7.84 6.8 21.6 F 7.95 7.2

10/3/10 1 1 22.6 8.21 7.3 22.8 7.96 6.9

10/4/10 1 2 24.2 8.19 6.2 350 24.2 8.06 6.7 346

10/5/10 1 3 24.6 8.14 7.5 24.6 8.20 8.7 24.5 8.32 7.5 25.0 8.67 8.2

10/6/10 1 4 24.2 8.21 7.6 24.1 8.28 7.6 24.1 8.28 7.8 24.7 8.42 8.7

10/7/10 1 5 25.7 8.05 7.9 25.6 8.35 7.8 25.5 8.35 8.0 26.0 8.63 8.3

10/8/10 1 6 24.2 8.20 7.4 24.3 8.22 7.5 24.2 8.24 7.8 25.0 8.49 9.4

10/9/10 1 7 25.2 8.18 7.5 25.2 8.27 7.6 25.2 8.16 7.8 25.6 8.49 8.6

10/10/10 1 8 24.3 8.19 7.4 24.2 8.19 7.4 24.4 8.17 7.6 24.7 8.45 8.5

10/11/10 1 9 25.5 8.04 7.0 365 25.4 8.11 7.5 362 25.5 8.20 8.0 361 26.0 8.46 9.3 359

10/12/10 2 0 25.5 8.31 8.0 25.6 8.33 8.0 25.6 8.41 8.3

10/13/10 2 1 25.0 8.10 7.0 24.9 8.18 7.3 24.7 8.24 7.8

10/14/10 2 2 25.6 8.09 7.7 25.5 8.16 7.8 25.6 8.26 8.4

10/15/10 2 3 25.3 8.36 8.0 25.3 8.62 8.3 25.3 8.59 8.6

10/16/10 2 4 24.0 8.25 7.3 24.5 8.15 8.2 24.4 7.9

10/17/10 2 5 25.3 8.05 7.5 25.5 8.12 8.0

10/18/10 2 6 24.4 8.03 7.2 356 24.7 7.81 8.0 360

10/19/10 2 7 24.1 8.07 8.4 24.2 8.26 8.4

10/20/10 2 8 24.3 8.10 7.1 355 24.3 8.22 8.0 359 8.34 8.5 355 8.33 8.5 360

DATE

TEST 

DAY

Replicate 1 Replicate 2 Replicate 3 Replicate 4
49

APPENDIX 5F. Water quality measurements Treatment F (nominal 15 mg TAN/L).

Temp pH DO SC Temp pH DO SC Temp pH DO SC Temp pH DO SC

9/22/10 0 25.8 8.09 418 25.8 8.05 7.1 419

9/23/10 1 25.7 25.7 7.86 6.9 7.97 6.9

9/24/10 2 26.1 7.96 6.4 410 26.1 8.03 6.5 410 25.9 8.02 6.5 420 26.0 8.03 6.5 417

9/25/10 3 23.8 7.98 6.6 24.1 7.99 6.9 24.4 8.05 6.9 24.2 8.02 6.8

9/26/10 4 24.2 8.04 7.3 24.2 8.04 7.4 24.2 7.98 7.1 24.0 8.03 6.9

9/27/10 5 24.0 8.10 7.1 399 23.8 8.07 7.1 422 23.8 8.00 7.0 418 24.2 8.04 7.2 424

9/28/10 6 25.5 7.5 25.7 8.22 7.7 25.3 8.21 7.4 25.7 8.20 7.5

9/29/10 7 8.22 24.7 8.05 7.4 24.0 8.00 7.3 24.7 8.01 7.8

9/30/10 8 25.1 8.04 7.4 25.0 8.08 7.5

10/1/10 9 23.2 7.80 6.4 23.3 7.43 3.3 F

10/2/10 1 0 22.3 8.02 7.5 21.7 8.02 7.4

10/3/10 1 1 22.7 8.33 7.9 22.6 8.07 7.9

10/4/10 1 2 24.3 7.99 7.2 414 24.3 8.09 6.6 428

10/5/10 1 3 24.6 8.23 7.8

10/6/10 1 4 24.2 8.26 7.9

10/7/10 1 5 25.5 8.29 8.0

10/8/10 1 6 24.3 8.11 7.2

10/9/10 1 7

10/10/10 1 8

10/11/10 1 9

10/12/10 2 0

10/13/10 2 1

10/14/10 2 2

10/15/10 2 3

10/16/10 2 4

10/17/10 2 5

10/18/10 2 6

10/19/10 2 7

10/20/10 2 8 8.30 8.9 410 8.21 8.5 412 8.25 8.5 413 8.18 8.4 413

DATE

TEST 

DAY

Replicate 1 Replicate 2 Replicate 3 Replicate 4
50

APPENDIX 6. TAN measurements.

Rep 1 Rep 2 Rep 3 Rep 4 Rep 1 Rep 2 Rep 3 Rep 4 Rep 1 Rep 2 Rep 3 Rep 4 Rep 1 Rep 2 Rep 3 Rep 4 Rep 1 Rep 2 Rep 3 Rep 4 Rep 1 Rep 2 Rep 3 Rep 4

9/22/10 0

9/23/10 1 0.060 0.060 0.060 0.060 1.219 1.295 1.404 1.528 2.695 2.854 2.558 2.188 3.739 4.048 4.048 3.943 6.960 7.338 7.233 7.061 10.782 11.033 10.982 10.932

9/24/10 2 0.060 0.060 0.060 0.060 0.504 0.440 0.373 0.237 0.611 1.101 1.077 0.719 2.084 1.815 2.590 2.820 3.998 3.854 4.224 4.147 10.446 10.961 9.781 11.009

9/25/10 3 0.060 0.060 0.060 0.060 0.750 0.720 1.418 1.835 1.964 3.619 3.769 8.617 9.077

9/26/10 4

9/27/10 5 0.060 0.060 0.060 0.060 0.144 0.199 0.206 0.210 0.499 0.493 0.512 1.078 1.400 1.420 1.334 3.238 3.471 3.602 3.553 8.797 9.337 9.084 9.337

9/28/10 6

9/29/10 7 0.060 0.060 0.111 0.150 0.545 0.572 1.358 0.729 3.433 3.576 8.392 8.852

9/30/10 8

10/1/10 9 0.060 0.060 0.340 0.099 0.747 0.842 0.728 2.316 4.766 4.961 10.515 9.131

10/2/10 10

10/3/10 11

10/4/10 12 0.060 0.060 0.060 0.060 0.060 0.060 0.275 0.308 1.469 1.593 11.006 9.350

10/5/10 13

10/6/10 14 0.060 0.060 0.121 0.138 0.711 1.170 1.502 5.298 5.418 14.791

10/7/10 15

10/8/10 16 0.060 0.060 0.060 0.060 0.146 0.060 0.476 0.231 0.879 0.331 1.214 2.303 2.938 1.721 2.104 5.699 5.809 5.536 5.837 18.841

10/9/10 17

10/10/10 18

10/11/10 19 0.060 0.060 0.060 0.060 0.824 0.291 0.825 0.821 1.624 0.913 2.554 1.655 2.878 3.172 3.285 3.809 6.107 6.317 6.570 7.183 16.369 16.369 14.198 16.152

10/12/10 20 0.060 0.529 0.060 0.552 0.060 1.130 0.912 1.166 1.687 4.783

10/13/10 21 0.060 0.060 0.060 0.060 0.416 0.060 0.402 0.339 1.131 0.738 0.907 2.000 2.307 2.200 4.636 4.879 4.811

10/14/10 22 0.060 0.060 0.060 0.060 0.363 0.327 0.390 0.994 1.112 0.353 0.596 2.263 2.239 1.907 4.440 4.637 4.597

10/15/10 23 0.060 0.060 0.060 0.060 0.475 0.501 0.517 0.468 1.271 0.427 1.211 2.569 2.689 1.259 4.937 5.344 5.194 11.890

10/16/10 24 0.060 0.060 0.060 0.060 0.472 0.496 0.511 0.508 1.245 0.348 0.771 2.383 2.414 2.262 4.278 4.598 10.008

10/17/10 25 0.060 0.060 0.060 0.060 0.489 0.541 0.566 0.607 1.305 0.060 0.604 2.312 2.322 2.312 4.362 4.613 11.355

10/18/10 26 0.060 0.060 0.060 0.060 0.403 0.530 0.523 0.452 1.171 0.060 0.248 1.984 2.011 1.984 4.011 4.337 10.057

10/19/10 27 0.060 0.060 0.060 0.060 0.484 0.543 0.454 0.432 1.047 0.557 0.671 2.127 2.221 2.197 4.367 4.650 12.170

10/20/10 28 0.060 0.060 0.060 0.060 0.545 0.739 0.468 0.471 1.185 0.241 0.953 0.657 1.979 2.005 1.936 2.238 4.011 4.152 4.206 4.665 9.972 10.058 10.015 10.101

D 7.5 mg TAN/L E 15 mg TAN/L

Date Test Day

Con A 0.9 mg TAN/L B 1.9 mg TAN/L C 3.8 mg TAN/L
51

APPENDIX 7. Trimatrix duplicate TAN analysis using Phenate Method.

SAMPLENAME SAMPNUM SAMPDATE STIME MATRIX REFER_CITATION PARAMETER CASNUM BATCH QCBATCH RUNDATE CHEM REPLIMIT RESULT UNIT Avg Dups GLEC T A % diff

8351 Con Rep3 1010329-01 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 <0.010 mg/L

8352 A 0.9mg/L Rep1 1010329-02 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.63 mg/L 0.529 0.84

8353 A 0.9mg/L Rep2 1010329-03 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.50 mg/L 0.50 ND

8375 A 0.9mg/L Rep2 DUP 1010329-04 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.50 mg/L

8354 A 0.9mg/L Rep3 1010329-05 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.71 mg/L 0.552 0.78

8355 A 0.9mg/L Rep4 1010329-06 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.57 mg/L ND

8356 B 1.9mg/L Rep1 1010329-07 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 1.3 mg/L 1.130 0.87

8357 B 1.9mg/L Rep2 1010329-08 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 1.1 mg/L 0.912 0.83

8359 B 1.9mg/L Rep4 1010329-09 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 1.4 mg/L 1.166 0.83

8360 C 3.8mg/L Rep4 1010329-10 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 2.0 mg/L 1.687 0.84

8361 D 7.5mg/L Rep2 1010329-11 10/12/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.050 5.3 mg/L 4.783 0.90

TML1 A 0.9mg/L Rep3 1010329-12 10/13/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.64 mg/L 0.402 0.63

TML2 B 1.9mg/L Rep2 1010329-13 10/13/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.9 mg/L 0.738 0.82

TML3 C 3.8mg/L Rep1 1010329-14 10/13/2010 16:45:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.020 2.6 mg/L 2.000 0.77

8716 Con Rep2 1010329-16 10/14/2010 13:58:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 <0.010 mg/L ND

8717 A 0.9mg/L Rep4 1010329-15 10/14/2010 13:54:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.57 mg/L 0.58 0.994 1.71

8718 A 0.9mg/LRep4Dup 1010329-17 10/14/2010 00:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0J18060 1010999 10/18/2010 GEH 0.010 0.59 mg/L

8720, Con 0.0 mg/LRep3 Dup 1010528-02 10/15/2010 13:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 <0.050 mg/L

8721, A 0.9mg/L Rep1 1010528-03 10/15/2010 13:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 0.70 mg/L 0.475 0.68

8722, B 1.9mg/L Rep 1 1010528-04 10/15/2010 13:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 1.7 mg/L 1.7 1.271 0.75

8723, B 1.9mg/L Rep 1 Dup 1010528-05 10/15/2010 13:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 1.7 mg/L

8724, C 3.8mg/L Rep2 1010528-06 10/15/2010 13:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.10 3 mg/L 2.95 2.689 0.91

8725, C 3.8mg/L Rep2 Dup 1010528-07 10/15/2010 13:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.10 2.9 mg/L

8732, A 0.9mg/L Rep1 1010528-08 10/18/2010 11:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 0.64 mg/L 0.635 0.403 0.63

8733, A 0.9mg/L Rep1 Dup 1010528-09 10/18/2010 11:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 0.63 mg/L

8734, B 1.9mg/L Rep 2 1010528-10 10/18/2010 11:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 <0.050 mg/L ND

8735, C 3.8mg/L Rep1 1010528-11 10/18/2010 11:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.10 2.6 mg/L 2.7 1.984 0.73

8736, C 3.8mg/L Rep1 Dup 1010528-12 10/18/2010 11:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.10 2.8 mg/L

8737, Con Rep 3 1010528-13 10/19/2010 14:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 <0.050 mg/L ND

8738, A 0.9mg/L Rep 3 1010528-14 10/19/2010 14:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 0.58 mg/L 0.57 0.454 0.80

8739, A 0.9mg/L Rep 3 Dup 1010528-15 10/19/2010 14:00:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 0.56 mg/L

8740, Con Rep 1 1010528-16 10/20/2010 11:30:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 <0.050 mg/L ND

8741, Con Rep 1 Dup 1010528-17 10/20/2010 11:30:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 <0.050 mg/L

8742, A 0.9mg/L Rep 4 1010528-18 10/20/2010 11:30:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.050 0.62 mg/L 0.471 0.76

8743, C 3.8mg/L Rep4 1010528-19 10/20/2010 11:30:00 Water SM 4500-NH3 G 20th Nitrogen, Ammonia 7664-41-7 0K02008 1011548 10/28/2010 GEH 0.10 3 mg/L 2.238 0.75

Equals Dups Avg % diff 0.78
52

APPENDIX 8A. P. canaliculata survival – control treatment through treatment B (nominal 1.9 mg TAN/L).

Rep 1 Rep 2 Rep 3 Rep 4 % Survival Rep 1 Rep 2 Rep 3 Rep 4 % Survival Rep 1 Rep 2 Rep 3 Rep 4 % Survival

9/22/10 0 6 6 6 6 100 6 6 6 6 100 6 6 6 6 100

9/23/10 1

9/24/10 2 6 5 6 6 96 5 6 6 6 96 6 6 3 3 75

9/25/10 3 6 5 6 5 92 5 5 6 6 92 6 6 1 1 58

9/26/10 4 6 5 6 5 92 5 5 6 6 92 6 6 0 1 54

9/27/10 5 6 5 6 5 92 5 5 6 6 92 6 6 0 1 54

9/28/10 6

9/29/10 7 6 5 6 5 92 5 5 6 6 92 6 5 0 1 50

9/30/10 8

10/1/10 9 6 5 6 5 92 5 5 6 6 92 6 5 0 1 50

10/2/10 10

10/3/10 11

10/4/10 12 6 5 6 4 88 5 5 6 6 92 6 4 0 1 46

10/5/10 13

10/6/10 14 6 5 6 4 88 5 5 6 6 92 6 2 0 1 38

10/7/10 15

10/8/10 16 6 5 6 4 88 5 5 6 6 92 6 2 0 1 38

10/9/10 17

10/10/10 18

10/11/10 19 6 5 6 4 88 5 5 6 6 92 6 1 0 1 33

10/12/10 20

10/13/10 21 5 5 6 4 83 5 5 6 6 92 6 1 0 1 33

10/14/10 22

10/15/10 23 5 5 6 4 83 5 5 6 6 92 6 1 0 1 33

10/16/10 24

10/17/10 25

10/18/10 26 5 5 6 4 83 5 5 6 6 92 6 1 0 1 33

10/19/10 27

10/20/10 28 5 5 6 4 83 5 5 6 6 92 6 1 0 1 33

10/21/10 29

10/22/10 30

10/23/10 31

10/24/10 32

10/25/10 33 5 5 6 4 83

10/26/10 34

10/27/10 35 5 5 6 4 83

10/28/10 36 5 5 6 4 83

10/29/10 37 5 5 6 4 83

10/30/10 38 5 5 6 4 83

10/31/10 39 5 5 6 4 83

11/1/10 40 5 5 6 3 79

11/2/10 41 5 5 6 3 79

11/3/10 42 5 4 6 3 75

Date Test Day

Con A 0.9mg/L B 1.9mg/L
53

APPENDIX 8B. P. canaliculata survival – treatments C through E (nominal 3.8 through 15 mg TAN/L).

Rep 1 Rep 2 Rep 3 Rep 4 % Survival Rep 1 Rep 2 Rep 3 Rep 4 % Survival Rep 1 Rep 2 Rep 3 Rep 4 % Survival

6 6 6 6 100 6 6 6 6 100 6 6 6 6 100

5 5 6 6 92 6 6 5 5 92 5 5 6 6 92

3 5 6 5 79 6 6 5 5 92 3 5 6 6 83

3 5 6 2 67 6 6 5 5 92 1 3 5 6 63

3 5 6 2 67 6 6 5 5 92 0 3 5 6 58

3 5 4 2 58 6 5 5 5 88 0 1 5 3 38

3 5 3 2 54 6 5 5 5 88 0 1 5 0 25

1 3 2 2 33 5 4 4 1 58 0 0 3 0 13

1 3 1 2 29 4 3 3 1 46 0 0 2 0 8

1 3 0 2 25 3 2 3 1 38 0 0 0 0

1 3 0 2 25 2 2 2 0 25 0 0 0 0

1 3 0 2 25 2 2 0 0 17 0 0 0 0

1 3 0 2 25 2 2 0 0 17 0 0 0 0

1 3 0 2 25 2 2 0 0 17 0 0 0 0

1 2 0 2 21 2 2 0 0 17 0 0 0 0

C 3.8mg/L D 7.5mg/L E 15mg/L
54

APPENDIX 9. P. canaliculata growth measurements.

Rep Means Treatment Means

Rep Snail #

Length

(cm)

Diameter

(cm)

Weight

(g)

Length

(mm)

Diameter

(cm)

Weight

(g)

Length

(mm)

Diameter

(cm)

Weight

(g)

A 1 1 0.694 0.227 1.23 Avg 0.78 0.34 1.62 0.80 0.39 1.9

A 1 2 0.906 0.419 2.37 Stdev 0.093 0.075 0.534 0.077 0.076 0.51

A 1 3 0.814 0.367 1.74 Avg +1 SD 0.87 0.42 2.15

A 1 4 0.802 0.381 1.75 Avg -1 SD 0.69 0.27 1.08

A 1 5 0.681 0.307 0.99

A 2 1 0.764 0.359 1.60 Avg 0.82 0.43 2.11

A 2 2 0.880 0.431 2.32 Stdev 0.058 0.044 0.38

A 2 3 0.756 0.456 1.81 Avg +1 SD 0.88 0.48 2.50

A 2 4 0.866 0.458 2.45 Avg -1 SD 0.76 0.39 1.73

A 2 5 0.844 0.465 2.39

B 1 1 1.032 0.422 2.56 Avg 0.84 0.40 1.9 0.82 0.39 1.8

B 1 2 0.965 0.459 2.66 Stdev 0.16 0.068 0.74 0.152 0.067 0.70

B 1 3 0.887 0.445 1.95 Avg +1 SD 1.00 0.47 2.62

B 1 4 0.811 0.438 2.04 Avg -1 SD 0.67 0.33 1.14

B 1 5 0.589 0.284 0.72

B 1 6 0.727 0.351 1.36

B 2 1 0.738 0.335 1.34 0.74 0.34 1.3

C1 1 0.949 0.444 2.80 0.95 0.44 2.8 0.87 0.41 2.1

0.119 0.072 0.87

C2 1 0.927 0.460 2.41 Avg 0.83 0.39 1.8

C2 2 0.732 0.328 1.14

D1 1 0.961 0.429 2.60 Avg 0.94 0.44 2.3 0.87 0.41 2.1

D1 2 0.926 0.444 1.93 0.088 0.030 0.38

D2 1 0.765 0.378 1.74 Avg 0.80 0.39 1.8

D2 2 0.838 0.400 1.94